Technical Field
[0001] The present invention relates to pharmaceutical compositions comprising an anti-IL-6
receptor antibody as an active ingredient, methods for producing the compositions,
and such.
Background Art
[0002] Antibodies are drawing attention as pharmaceuticals as they are highly stable in
plasma (blood) and have few adverse effects. Of them, a number of IgG-type antibody
pharmaceuticals are available on the market and many antibody pharmaceuticals are
currently under development (Non-patent Documents 1 and 2).
[0003] IL-6 is a cytokine involved in various autoimmune diseases (Non-patent Document 3).
It is thought that TOCILIZUMAB, a humanized anti-IL-6 receptor IgG1 antibody, can
be useful as a therapeutic agent for IL-6-associated diseases such as rheumatoid arthritis,
since it specifically binds to the IL-6 receptor and thereby neutralizes its biological
activity (Patent Documents 1 to 3 and Non-patent Document 4). In fact, TOCILIZUMAB
has been approved as a therapeutic agent for Castleman's disease in Japan (Non-patent
Document 5).
[0004] Various technologies applicable to second-generation antibody pharmaceuticals have
been developed, including those that enhance effector function, antigen-binding activity,
retention in plasma (blood), or stability, and those that reduce the risk of immunogenicity
(antigenicity).
[0005] For methods of enhancing drug efficacy or reducing dosage, technologies that enhance
antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity
(CDC) through amino acid substitution in the Fc domain of an IgG antibody have been
reported (Non-patent Document 6). Furthermore, affinity maturation has been reported
as a technology for enhancing antigen-binding activity or antigen-neutralizing activity
(Non-patent Document 7). This technology enables enhancement of antigen-binding activity
through introduction of amino acid mutations into the CDR region of a variable region
or such. The enhancement of antigen-binding activity enables to improve
in vitro biological activity or reduce dosage, and further to improve
in vivo efficacy (Non-patent Document 8). Currently, clinical trials are being conducted
to assess Motavizumab (produced by affinity maturation), which is expected to have
a superior effect than Palivizumab (a first generation pharmaceutical of anti-RSV
antibody) (Non-patent Document 9). Alternatively, a more superior effect can be produced
by binding to a different epitope. For example, it has been reported that Oftamumab
which recognizes an epitope different from that recognized by Rituximab (an anti-CD20
antibody) shows a more superior effect
in vivo than Rituximab. Currently, clinical trials are being conducted to assess Oftamumab
(Non-patent Document 10). There is no previous report describing a human, humanized,
or chimeric anti-IL-6 receptor antibody having an affinity greater than 1 nM.
[0006] A problem encountered with current antibody pharmaceuticals is high production cost
associated with the administration of extremely large quantities of protein. For example,
the dosage of TOCILIZUMAB, a humanized anti-IL-6 receptor IgG1 antibody, has been
estimated to be about 8 mg/kg/month by intravenous injection (Non-patent Document
4). Its preferred form of administration is thought to be subcutaneous formulation
in chronic autoimmune diseases. In general, it is necessary that subcutaneous formulations
are high concentration formulations. From the perspective of stability or such, the
concentration limit for IgG-type antibody formulations is in general thought to be
about 100 mg/ml (Non-patent Document 11). Low-cost, convenient second-generation antibody
pharmaceuticals that can be administered subcutaneously in longer intervals can be
provided by increasing the half-life of an antibody in the plasma to prolong its therapeutic
effect and thereby reduce the amount of protein administered, and by conferring the
antibody with high stability so that high concentration formulations can be prepared.
[0007] A possible method for improving antibody half-life in the plasma has been reported,
and it artificially substitutes amino acids in the constant region (Non-patent Documents
12 and 13). However, introduction of non-natural sequences into the constant region
is not preferred from the perspective of immunogenicity risk. Although it is preferable
to introduce amino acid substitutions into the variable region rather than to the
constant region from the perspective of immunogenicity, there is no report on improvement
of antibody half-life in the plasma by substituting amino acids in the variable region,
or improvement of the half-life of a human, humanized, or chimeric IL-6 receptor antibody
in the plasma.
[0008] Methods for improving stability have been reported, and these include amino acid
substitution or shuffling in the framework of the variable region to improve physicochemical
stability (intermediate temperature of thermal denaturation) (Non-patent Documents
14 and 15). However, there is no previous report suggesting that such amino acid substitution
improves the stability (suppresses the aggregation) in formulations that have a concentration
higher than 100 mg/ml. There is also no previous report on stable human or humanized
IL-6 receptor antibody molecules in formulations of higher-than-100 mg/ml concentrations.
[0009] Another important problem encountered in developing biopharmaceuticals is immunogenicity.
In general, the immunogenicity of mouse antibodies is reduced by antibody humanization.
It is assumed that immunogenicity risk can be further reduced by using a germline
framework sequence as a template in antibody humanization (Non-patent Document 16).
However, even Adalimumab, a fully human anti-TNF antibody, showed high-frequency (13%
to 17%) immunogenicity, and the therapeutic effect was found to be reduced in patients
who showed immunogenicity (Non-patent Documents 17 and 18). T-cell epitopes may be
present even in the CDR regions of human antibodies, and these T-cell epitopes in
CDR are a possible cause of immunogenicity.
In silico and
in vivo methods for predicting T-cell epitopes have been reported (Non-patent Documents 19
and 20). It is assumed that immunogenicity risk can be reduced by removing T-cell
epitopes predicted using such methods (Non-patent Document 21). TOCILIZUMAB is a humanized
anti-IL-6 receptor IgG1 antibody obtained by humanizing a mouse PM-1 antibody. The
antibody is yielded by CDR grafting using human NEW and REI framework sequences as
template for H and L chains, respectively; however, a five-amino acid mouse sequence
is retained in the antibody as framework and it is essential for retaining the antibody
activity (Non-patent Document 22). There is no previous report on the substitution
of a human sequence for the mouse sequence that remains in the framework of the humanized
antibody TOCILIZUMAB, without loss of the activity. Furthermore, the CDR sequence
of TOCILIZUMAB is a mouse sequence. Thus, there is a possibility that, like Adalimumab,
TOCILIZUMAB contains T-cell epitopes in its CDR region, and may have a potential immunogenicity
risk. Furthermore, TOCILIZUMAB belongs to the IgG1 subclass, and therefore can bind
to Fcγ receptor. Since Fcγ receptor is expressed in antigen-presenting cells, molecules
that bind to Fcγ receptor tend to be presented as antigens. It has been reported that
immunogenicity is and can be enhanced by linking a protein or peptide to the Fc domain
of IgG1 (Non-patent Document 23 and Patent Document 4). In clinical trials of TOCILIZUMAB,
antibodies against TOCILIZUMAB were not detected when TOCILIZUMAB was used at an effective
dose (8 mg/kg). However, immunogenicity was observed at the doses of 2 and 4 mg/kg
(Patent Document 5). This suggests that the immunogenicity of the humanized anti-IL-6
receptor IgG1 antibody TOCILIZUMAB can be reduced. However, there has been no report
on reducing the immunogenicity risk of TOCILIZUMAB (a humanized PM-1 antibody) by
amino acid substitution.
[0010] In recent years, the safety of antibody pharmaceuticals has become of great importance.
Interaction between the antibody Fc domain and Fcγ receptor is assumed to be a cause
of serious adverse effect encountered in phase-I clinical trials of TGN1412 (Non-patent
Document 24). In antibody pharmaceuticals aimed at neutralizing the biological activity
of an antigen, binding of the Fc domain to Fcγ receptor, which is important for the
effector function such as ADCC, is unnecessary. In addition, as described above, the
binding to Fcγ receptor may be unfavorable from the perspective of immunogenicity
and adverse effects.
[0011] A method for impairing the binding to Fcγ receptor is to alter the isotype of the
IgG antibody from IgG1 to IgG2 or IgG4; however, this method cannot completely inhibit
the binding (Non-patent Document 25). One of the methods reported for completely inhibiting
the binding to Fcγ receptor is to artificially alter the Fc domain. For example, the
effector functions of anti-CD3 antibodies and anti-CD4 antibodies cause adverse effects.
Thus, amino acids that are not present in the wild type sequence were introduced into
the Fcγ receptor-binding domain of Fc (Non-patent Documents 26 and 27), and clinical
trials are currently being conducted to assess anti-CD3 antibodies that do not bind
to Fcγ receptor and anti-CD4 antibodies that have a mutated Fc domain (Non-patent
Documents 24 and 28). Alternatively, Fcγ receptor-nonbinding antibodies can be prepared
by altering the FcγR-binding domain of IgG1 (at positions 233, 234, 235, 236, 327,
330, and 331 in the EU numbering system) to an IgG2 or IgG4 sequence (Non-patent Document
29 and Patent Document 6). However, these molecules contain novel non-natural peptide
sequences of nine to twelve amino acids, which may constitute a T-cell epitope peptide
and thus pose immunogenicity risk. There is no previous report on Fcγ receptor-nonbinding
antibodies that have overcome these problems.
[0012] Meanwhile, physicochemical properties of antibody proteins, in particular, homogeneity
and stability, are very crucial in the development of antibody pharmaceuticals. For
the IgG2 isotype, significant heterogeneity derived from disulfide bonds in the hinge
region has been reported (Non-patent Document 30). It is not easy to manufacture them
as a pharmaceutical in large-scale while maintaining the objective substances/related
substances related heterogeneity derived from disulfide bonds between productions.
Thus, single substances are desirable as much as possible for antibody molecules developed
as pharmaceuticals.
[0013] IgG2 and IgG4 are unstable under acidic conditions. IgG type antibodies are in general
exposed to acidic conditions in the purification process using Protein A and the virus
inactivation process. Thus, there is a possibility that IgG2 and IgG4 undergo denaturation
and aggregation during these processes. It is thus preferred that antibody molecules
developed as pharmaceuticals are also stable under acidic conditions. Natural IgG2
and IgG4, and Fcγ receptor-nonbinding antibodies derived from IgG2 or IgG4 (Non-patent
Documents 25 and 26 and Patent Document 6) have such problems. It is desirable to
solve these problems when developing antibodies into pharmaceuticals.
[0014] IgG1-type antibodies are relatively stable under acidic conditions, and the degree
of heterogeneity originated from disulfide bonds in the hinge region is also lower
in this type of antibodies. However, IgG1-type antibodies are reported to undergo
non-enzymatic peptide bond cleavage in the hinge region in solutions when they are
stored as formulations, and Fab fragments are generated as impurities as a result
(Non-patent Document 31). It is desirable to overcome the generation of impurity when
developing antibodies into pharmaceuticals.
[0015] Furthermore, for heterogeneity of the C-terminal sequences of an antibody, deletion
of C-terminal amino acid lysine residue, and amidation of the C-terminal amino group
due to deletion of both of the two C-terminal amino acids, glycine and lysine, have
been reported (Non-patent Document 32). It is preferable to eliminate such heterogeneity
when developing antibodies into pharmaceuticals.
[0016] The constant region of an antibody pharmaceutical aimed for neutralizing an antigen
preferably has a sequence that overcomes all the problems described above. However,
a constant region that meets all the requirements has not been reported.
[0017] There is no previous report on the development of second-generation molecules that
exhibit an improved antigen-neutralizing activity and produce a prolonged therapeutic
effect even when the frequency of administration is reduced, and which have reduced
immunogenicity and improved safety and physicochemical properties, as compared to
first-generation molecules. There is also no report on second-generation TOCILIZUMAB,
which has more superiority in terms of the requirements described above by altering
amino acid sequences of the variable and constant regions of the humanized anti-IL-6
receptor IgG1 antibody TOCILIZUMAB.
[0018] Documents of related prior arts for the present invention are described below.
[Non-patent Document 1] Janice M Reichert, Clark J Rosensweig, Laura B Faden & Matthew C Dewitz. Monoclonal
antibody successes in the clinic. Nature Biotechnology (2005) 23, 1073-1078
[Non-patent Document 2] Pavlou AK, Belsey MJ. The therapeutic antibodies market to 2008. Eur. J. Pharm. Biopharm.
2005 Apr;59(3):389-96
[Non-patent Document 3] Nishimoto N, Kishimoto T. Interleukin 6: from bench to bedside. Nat. Clin. Pract.
Rheumatol. 2006 Nov;2(11):619-26
[Non-patent Document 4] Maini RN, Taylor PC, Szechinski J, Pavelka K, Broll J, Balint G, Emery P, Raemen F,
Petersen J, Smolen J, Thomson D, Kishimoto T, CHARISMA Study Group. Double-blind randomized
controlled clinical trial of the interleukin-6 receptor antagonist, Tocilizumab, in
European patients with rheumatoid arthritis who had an incomplete response to methotrexate.
Arthritis Rheum. 2006 Sep;54(9):2817-29
[Non-patent Document 5] Nishimoto N, Kanakura Y, Aozasa K, Johkoh T, Nakamura M, Nakano S, Nakano N, Ikeda
Y, Sasaki T, Nishioka K, Hara M, Taguchi H, Kimura Y, Kato Y, Asaoku H, Kumagai S,
Kodama F, Nakahara H, Hagihara K, Yoshizaki K, Kishimoto T. Humanized anti-interleukin-6
receptor antibody treatment of multicentric Castleman disease. Blood 2005 Oct 15;106(8):2627-32
[Non-patent Document 6] Kim SJ, Park Y, Hong HJ. Antibody engineering for the development of therapeutic antibodies.
Mol. Cells 2005 Aug 31;20(1):17-29 Review
[Non-patent Document 7] Rothe A, Hosse RJ, Power BE. Ribosome display for improved biotherapeutic molecules.
Expert. Opin. Biol. Ther. 2006 Feb;6(2):177-87
[Non-patent Document 8] Rajpal A, Beyaz N, Haber L, Cappuccilli G, Yee H, Bhatt RR, Takeuchi T, Lerner RA,
Crea R. A general method for greatly improving the affinity of antibodies by using
combinatorial libraries. Proc. Natl. Acad. Sci. USA. 2005 Jun 14;102(24):8466-71.
Epub. 2005 Jun 6
[Non-patent Document 9] Wu H, Pfarr DS, Johnson S, Brewah YA, Woods RM, Patel NK, White WI, Young JF, Kiener
PA. Development of Motavizumab, an Ultra-potent Antibody for the Prevention of Respiratory
Syncytial Virus Infection in the Upper and Lower Respiratory Tract. J. Mol. Biol,
(2007) 368, 652-665
[Non-patent Document 10] Teeling JL, Mackus WJ, Wiegman LJ, van den Brakel JH, Beers SA, French RR, van Meerten
T, Ebeling S, Vink T, Slootstra JW, Parren PW, Glennie MJ, van de Winkel JG. The biological
activity of human CD20 monoclonal antibodies is linked to unique epitopes on CD20.
J. Immunol. 2006 Jul 1;177(1):362-71
[Non-patent Document 11] Shire SJ, Shahrokh Z, Liu J. Challenges in the development of high protein concentration
formulations. J. Pharm. Sci. 2004 Jun;93(6):1390-402
[Non-patent Document 12] Hinton PR, Xiong JM, Johlfs MG, Tang MT, Keller S, Tsurushita N. An engineered human
IgG1 antibody with longer serum half-life. J. Immunol. 2006 Jan 1;176(1):346-56
[Non-patent Document 13] Ghetie V, Popov S, Borvak J, Radu C, Matesoi D, Medesan C, Ober RJ, Ward ES. Increasing
the serum persistence of an IgG fragment by random mutagenesis. Nat. Biotechnol. 1997
Jul;15(7):637-40
[Non-patent Document 14] Ewert S, Honegger A, Pluckthun A. Stability improvement of antibodies for extracellular
and intracellular applications: CDR grafting to stable frameworks and structure-based
framework engineering. Methods. 2004 Oct;34(2):184-99 Review [Non-patent Document 15] Damschroder MM, Widjaja L, Gill PS, Krasnoperov V, Jiang W, Dall'Acqua WF, Wu H. Framework
shuffling of antibodies to reduce immunogenicity and manipulate functional and biophysical
properties. Mol. Immunol. 2007 Apr;44(11):3049-60 [Non-patent Document 16] Hwang WY, Almagro JC, Buss TN, Tan P, Foote J. Use of human germline genes in a CDR
homology-based approach to antibody humanization. Methods. 2005 May;36(1):35-42
[Non-patent Document 17] Bartelds GM, Wijbrandts CA, Nurmohamed MT, Stapel S, Lems WF, Aarden L, Dijkmans BA,
Tak P, Wolbink GJ. Clinical response to adalimumab: The relationship with anti-adalimumab
antibodies and serum adalimumab concentrations in rheumatoid arthritis. Ann. Rheum.
Dis. Jul;66(7):921-6 Epub 2007 Feb 14
[Non-patent Document 18] Bender NK, Heilig CE, Droll B, Wohlgemuth J, Armbruster FP, Heilig B. Immunogenicity,
efficacy and adverse events of adalimumab in RA patients. Rheumatol. Int. 2007 Jan;27(3):269-74
[Non-patent Document 19] Van Walle I, Gansemans Y, Parren PW, Stas P, Lasters I. Immunogenicity screening in
protein drug development. Expert Opin. Biol. Ther. 2007 Mar;7(3):405-18
[Non-patent Document 20] Jones TD, Phillips WJ, Smith BJ, Bamford CA, Nayee PD, Baglin TP, Gaston JS, Baker
MP. Identification and removal of a promiscuous CD4+ T cell epitope from the Cl domain
of factor VIII. J. Thromb. Haemost. 2005 May;3(5):991-1000
[Non-patent Document 21] Chirino AJ, Ary ML, Marshall SA. Minimizing the immunogenicity of protein therapeutics.
Drug Discov. Today 2004 Jan 15;9(2):82-90
[Non-patent Document 22] Sato K, Tsuchiya M, Saldanha J, Koishihara Y, Ohsugi Y, Kishimoto T, Bendig MM. Reshaping
a human antibody to inhibit the interleukin 6-dependent tumor cell growth. Cancer
Res. 1993 Feb 15;53(4):851-6
[Non-patent Document 23] Guyre PM, Graziano RF, Goldstein J, Wallace PK, Morganelli PM, Wardwell K, Howell
AL. Increased potency of Fc-receptor-targeted antigens. Cancer Immunol. Immunother.
1997 Nov-Dec;45(3-4):146-8
[Non-patent Document 24] Strand V, Kimberly R, Isaacs JD. Biologic therapies in rheumatology: lessons learned,
future directions. Nat. Rev. Drug Discov. 2007 Jan;6(1):75-92 [Non-patent Document 25] Gessner JE, Heiken H, Tamm A, Schmidt RE. The IgG Fc receptor family. Ann. Hematol.
1998 Jun;76(6):231-48
[Non-patent Document 26] Cole MS, Anasetti C, Tso JY. Human IgG2 variants of chimeric anti-CD3 are nonmitogenic
to T cells. J. Immunol. 1997 Oct 1;159(7):3613-21
[Non-patent Document 27] Reddy MP, Kinney CA, Chaikin MA, Payne A, Fishman-Lobell J, Tsui P, Dal Monte PR,
Doyle ML, Brigham-Burke MR, Anderson D, Reff M, Newman R, Hanna N, Sweet RW, Truneh
A. Elimination of Fc receptor-dependent effector functions of a modified IgG4 monoclonal
antibody to human CD4. J. Immunol. 2000 Feb 15; 164(4): 1925-33
[Non-patent Document 28] Chau LA, Tso JY, Melrose J, Madrenas J. HuM291(Nuvion), a humanized Fc receptor-nonbinding
antibody against CD3, anergizes peripheral blood T cells as partial agonist of the
T cell receptor. Transplantation 2001 Apr 15;71(7):941-50
[Non-patent Document 29] Armour KL, Clark MR, Hadley AG, Williamson LM. Recombinant human IgG molecules lacking
Fcgamma receptor I binding and monocyte triggering activities. Eur. J. Immunol. 1999
Aug;29(8):2613-24
[Non-patent Document 30] Chu GC, Chelius D, Xiao G, Khor HK, Coulibaly S, Bondarenko PV. Accumulation of Succinimide
in a Recombinant Monoclonal Antibody in Mildly Acidic Buffers Under Elevated Temperatures.
Pharm. Res. 2007 Mar 24;24(6):1145-56
[Non-patent Document 31] AJ Cordoba, BJ Shyong, D Breen, RJ Harris. Nonenzymatic hinge region fragmentation
of antibodies in solution. J. Chromatogr. B. Anal. Technol. Biomed. Life Sci. (2005)
818, 115-121
[Non-patent Document 32] Johnson KA, Paisley-Flango K, Tangarone BS, Porter TJ, Rouse JC. Cation exchange-HPLC
and mass spectrometry reveal C-terminal amidation of an IgG1 heavy chain. Anal. Biochem.
2007 Jan 1;360(1):75-83
[Patent Document 1] WO 92/19759
[Patent Document 2] WO 96/11020
[Patent Document 3] WO 96/12503
[Patent Document 4] US 20050261229A1
[Patent Document 5] WO 2004096273 (A1)
[Patent Document 6] WO 99/58572
Disclosure of the Invention
[Problems to be Solved by the Invention]
[0019] The present invention was achieved in view of the above circumstances. An objective
of the present invention is to provide pharmaceutical compositions that comprise second-generation
molecules, which are more superior than the humanized anti-IL-6 receptor IgG 1 antibody
TOCILIZUMAB, and methods for producing such pharmaceutical compositions. The second-generation
molecules have been improved to exhibit enhanced antigen-neutralizing activity and
pharmacokinetics (retention in plasma), and thus produce a prolonged therapeutic effect
even when the frequency of administration is reduced; and they have also been improved
to have reduced immunogenicity and improved safety and physicochemical properties,
by altering amino acid sequences of the variable and constant regions of TOCILIZUMAB.
[Means for Solving the Problems]
[0020] The present inventors conducted dedicated studies to generate second-generation molecules
that are more superior than the first-generation humanized anti-IL-6 receptor IgG1
antibody TOCILIZUMAB, and have been improved to exhibit enhanced drug efficacy and
pharmacokinetics, and thus produce a prolonged therapeutic effect even when the frequency
of administration is reduced. They have also been improved to have reduced immunogenicity
and improved safety and physicochemical properties (stability and homogeneity), by
altering amino acid sequences of the variable and constant regions of TOCILIZUMAB.
As a result, the present inventors discovered multiple CDR mutations in the variable
regions OF TOCILIZUMAB that enable to improve the antigen-binding activity (affinity).
The present inventors thus successfully improved the affinity significantly using
a combination of such mutations. The present inventors also successfully improved
pharmacokinetics by altering the variable region sequence to lower the isoelectric
point of an antibody. Furthermore, the present inventors successfully reduced immunogenicity
risk by removing some of the
in silico-predicted T-cell epitope peptides in the variable regions and the mouse sequences
that remain in the framework of TOCILIZUMAB. In addition, the present inventors successfully
increased the stability at higher concentrations. Furthermore, the present inventors
also successfully discovered novel constant region sequences that do not bind to Fcγ
receptor and that improve the stability under acidic conditions, heterogeneity originated
from disulfide bonds in the hinge region, heterogeneity originated from the H-chain
C terminus, and stability in high concentration formulations, while minimizing the
generation of new T-cell epitope peptides in the constant region of TOCILIZUMAB. The
present inventors successfully discovered second-generation molecules that are more
superior to TOCILIZUMAB, by combining amino acid sequence alterations in the CDR,
variable, and constant regions.
[0021] The present invention relates to pharmaceutical compositions comprising a humanized
anti-IL-6 receptor IgG1 antibody that has been improved to exhibit more superior antigen
(IL-6 receptor)-binding activity, more prolonged retention in plasma, more excellent
safety and physicochemical properties (stability and homogeneity), and further reduced
immunogenicity risk, by altering the amino acid sequences of variable and constant
regions of the humanized anti-IL-6 receptor TgG1 antibody TOCILIZUMAB; and methods
for producing such pharmaceutical compositions. More specifically, the present invention
provides:
[1] an anti-IL-6 receptor antibody of any one of:
- (a) an antibody that comprises a heavy chain variable region comprising CDR1 in which
Ser at position 1 in the amino acid sequence of SEQ ID NO: 1 has been substituted
with another amino acid;
- (b) an antibody that comprises a heavy chain variable region comprising CDR1 in which
Trp at position 5 in the amino acid sequence of SEQ ID NO: 1 has been substituted
with another amino acid;
- (c) an antibody that comprises a heavy chain variable region comprising CDR2 in which
Tyr at position 1 in the amino acid sequence of SEQ ID NO: 2 has been substituted
with another amino acid;
- (d) an antibody that comprises a heavy chain variable region comprising CDR2 in which
Thr at position 8 in the amino acid sequence of SEQ ID NO: 2 has been substituted
with another amino acid;
- (e) an antibody that comprises a heavy chain variable region comprising CDR2 in which
Thr at position 9 in the amino acid sequence of SEQ ID NO: 2 has been substituted
with another amino acid;
- (f) an antibody that comprises a heavy chain variable region comprising CDR3 in which
Ser at position 1 in the amino acid sequence of SEQ ID NO: 3 has been substituted
with another amino acid;
- (g) an antibody that comprises a heavy chain variable region comprising CDR3 in which
Leu at position 2 in the amino acid sequence of SEQ ID NO: 3 has been substituted
with another amino acid;
- (h) an antibody that comprises a heavy chain variable region comprising CDR3 in which
Thr at position 5 in the amino acid sequence of SEQ ID NO: 3 has been substituted
with another amino acid;
- (i) an antibody that comprises a heavy chain variable region comprising CDR3 in which
Ala at position 7 in the amino acid sequence of SEQ ID NO: 3 has been substituted
with another amino acid;
- (j) an antibody that comprises a heavy chain variable region comprising CDR3 in which
Met at position 8 in the amino acid sequence of SEQ ID NO: 3 has been substituted
with another amino acid;
- (k) an antibody that comprises a heavy chain variable region comprising CDR3 in which
Ser at position 1 and Thr at position 5 in the amino acid sequence of SEQ ID NO: 3
have been substituted with other amino acids;
- (l) an antibody that comprises a heavy chain variable region comprising CDR3 in which
Leu at position 2, Ala at position 7, and Met at position 8 in the amino acid sequence
of SEQ ID NO: 3 have been substituted with other amino acids;
- (m) an antibody that comprises a light chain variable region comprising CDR1 in which
Arg at position 1 in the amino acid sequence of SEQ ID NO: 4 has been substituted
with another amino acid;
- (n) an antibody that comprises a light chain variable region comprising CDR1 in which
Gln at position 4 in the amino acid sequence of SEQ ID NO: 4 has been substituted
with another amino acid;
- (o) an antibody that comprises a light chain variable region comprising CDR1 in which
Tyr at position 9 in the amino acid sequence of SEQ ID NO: 4 has been substituted
with another amino acid;
- (p) an antibody that comprises a light chain variable region comprising CDR1 in which
Asn at position 11 in the amino acid sequence of SEQ ID NO: 4 has been substituted
with another amino acid;
- (q) an antibody that comprises a light chain variable region comprising CDR2 in which
Thr at position 2 in the amino acid sequence of SEQ ID NO: 5 has been substituted
with another amino acid;
- (r) an antibody that comprises a light chain variable region comprising CDR3 in which
Gln at position 1 in the amino acid sequence of SEQ ID NO: 6 has been substituted
with another amino acid;
- (s) an antibody that comprises a light chain variable region comprising CDR3 in which
Gly at position 3 in the amino acid sequence of SEQ ID NO: 6 has been substituted
with another amino acid;
- (t) an antibody that comprises a light chain variable region comprising CDR1 in which
Tyr at position 9 in the amino acid sequence of SEQ ID NO: 4 has been substituted
with another amino acid, and CDR3 in which Gly at position 3 in the amino acid sequence
of SEQ ID NO: 6 has been substituted with another amino acid;
- (u) an antibody that comprises a light chain variable region comprising CDR3 in which
Thr at position 5 in the amino acid sequence of SEQ ID NO: 6 has been substituted
with another amino acid;
- (v) an antibody that comprises a light chain variable region comprising CDR3 in which
Gln at position 1 and Thr at position 5 in the amino acid sequence of SEQ ID NO: 6
have been substituted with other amino acids;
- (w) an antibody that comprises a heavy chain variable region comprising CDR2 in which
Thr at position 9 in the amino acid sequence of SEQ ID NO: 2 has been substituted
with another amino acid, and CDR3 in which Ser at position 1 and Thr at position 5
in the amino acid sequence of SEQ ID NO: 3 have been substituted with other amino
acids;
- (x) an antibody that comprises the heavy chain variable region of (k) and the light
chain variable region of (v); and
- (y) the antibody of (x) that further comprises the CDR2 of (e);
[2] an anti-IL-6 receptor antibody that comprises a light chain variable region comprising
CDR2 in which Thr at position 2 in the amino acid sequence of SEQ ID NO: 5 has been
substituted with another amino acid;
[3] an anti-IL-6 receptor antibody of any one of:
- (a) an antibody that comprises a heavy chain variable region comprising FR1 in which
Arg at position 13 in the amino acid sequence of SEQ ID NO: 7 has been substituted
with another amino acid;
- (b) an antibody that comprises a heavy chain variable region comprising FR1 in which
Gln at position 16 in the amino acid sequence of SEQ ID NO: 7 has been substituted
with another amino acid;
- (c) an antibody that comprises a heavy chain variable region comprising FR1 in which
Thr at position 23 in the amino acid sequence of SEQ ID NO: 7 has been substituted
with another amino acid;
- (d) an antibody that comprises a heavy chain variable region comprising FR1 in which
Thr at position 30 in the amino acid sequence of SEQ ID NO: 7 has been substituted
with another amino acid;
- (e) an antibody that comprises a heavy chain variable region comprising FR1 in which
Arg at position 13, Gln at position 16, Thr at position 23, and Thr at position 30
in the amino acid sequence of SEQ ID NO: 7 have been substituted with other amino
acids;
- (f) an antibody that comprises a heavy chain variable region comprising FR2 in which
Arg at position 8 in the amino acid sequence of SEQ ID NO: 8 has been substituted
with another amino acid;
- (g) an antibody that comprises a heavy chain variable region comprising FR3 in which
Met at position 4 in the amino acid sequence of SEQ ID NO: 9 has been substituted
with another amino acid;
- (h) an antibody that comprises a heavy chain variable region comprising FR3 in which
Leu at position 5 in the amino acid sequence of SEQ ID NO: 9 has been substituted
with another amino acid;
- (i) an antibody that comprises a heavy chain variable region comprising FR3 in which
Arg at position 16 in the amino acid sequence of SEQ ID NO: 9 has been substituted
with another amino acid;
- (j) an antibody that comprises a heavy chain variable region comprising FR3 in which
Val at position 27 in the amino acid sequence of SEQ ID NO: 9 has been substituted
with another amino acid;
- (k) an antibody that comprises a heavy chain variable region comprising FR3 in which
Met at position 4, Leu at position 5, Arg at position 16, and Val at position 27 in
the amino acid sequence of SEQ ID NO: 9 have been substituted with other amino acids;
- (l) an antibody that comprises a heavy chain variable region comprising FR4 in which
Gln at position 3 in the amino acid sequence of SEQ ID NO: 10 has been substituted
with another amino acid;
- (m) an antibody that comprises a light chain variable region comprising FR1 in which
Arg at position 18 in the amino acid sequence of SEQ ID NO: 11 has been substituted
with another amino acid;
- (n) an antibody that comprises a light chain variable region comprising FR2 in which
Lys at position 11 in the amino acid sequence of SEQ ID NO: 12 has been substituted
with another amino acid;
- (o) an antibody that comprises a light chain variable region comprising FR3 in which
Gln at position 23 in the amino acid sequence of SEQ ID NO: 13 has been substituted
with another amino acid;
- (p) an antibody that comprises a light chain variable region comprising FR3 in which
Pro at position 24 in the amino acid sequence of SEQ ID NO: 13 has been substituted
with another amino acid;
- (q) an antibody that comprises a light chain variable region comprising FR3 in which
Ile at position 27 in the amino acid sequence of SEQ ID NO: 13 has been substituted
with another amino acid;
- (r) an antibody that comprises a light chain variable region comprising FR3 in which
Gln at position 23, Pro at position 24, and Ile at position 27 in the amino acid sequence
of SEQ ID NO: 13 have been substituted with other amino acids:
- (s) an antibody that comprises a light chain variable region comprising FR4 in which
Lys at position 10 in the amino acid sequence of SEQ ID NO: 14 has been substituted
with another amino acid;
- (t) an antibody that comprises a heavy chain variable region comprising FR4 in which
Ser at position 5 in the amino acid sequence of SEQ ID NO: 10 has been substituted
with another amino acid;
- (u) an antibody that comprises a heavy chain variable region comprising FR4 in which
Gln at position 3 and Ser at position 5 in the amino acid sequence of SEQ ID NO: 10
have been substituted with other amino acids;
- (v) an antibody that comprises a heavy chain variable region comprising FR3 comprising
the amino acid sequence of SEQ ID NO: 184;
- (w) an antibody that comprises a heavy chain variable region comprising the FR1 of
(e), FR2 of (f), FR3 of (k), and FR4 of (l) or (u);
- (x) an antibody that comprises a light chain variable region comprising the FR1 of
(m), FR2 of (n), FR3 of (r), and FR4 of (s); and
- (y) an antibody that comprises the heavy chain variable region of (w) and the light
chain variable region of (x);
[4] an anti-IL-6 receptor antibody of any one of:
- (a) an antibody that comprises a heavy chain variable region comprising CDR1 in which
Ser at position 1 in the amino acid sequence of SEQ ID NO: 1 has been substituted
with another amino acid;
- (b) an antibody that comprises a heavy chain variable region comprising CDR2 in which
Thr at position 9 in the amino acid sequence of SEQ ID NO: 2 has been substituted
with another amino acid;
- (c) an antibody that comprises a heavy chain variable region comprising CDR2 in which
Ser at position 16 in the amino acid sequence of SEQ ID NO: 2 has been substituted
with another amino acid;
- (d) an antibody that comprises a heavy chain variable region comprising CDR2 in which
Thr at position 9 and Ser at position 16 in the amino acid sequence of SEQ ID NO:
2 have been substituted with other amino acids;
- (e) an antibody that comprises a light chain variable region comprising CDR1 in which
Arg at position 1 in the amino acid sequence of SEQ ID NO: 4 has been substituted
with another amino acid;
- (f) an antibody that comprises a light chain variable region comprising CDR2 in which
Thr at position 2 in the amino acid sequence of SEQ ID NO: 5 has been substituted
with another amino acid;
- (g) an antibody that comprises a light chain variable region comprising CDR2 in which
Arg at position 4 in the amino acid sequence of SEQ ID NO: 5 has been substituted
with another amino acid;
- (h) an antibody that comprises a light chain variable region comprising CDR2 in which
Thr at position 2 and Arg at position 4 in the amino acid sequence of SEQ ID NO: 5
have been substituted with other amino acids;
- (i) an antibody that comprises a light chain variable region comprising CDR3 in which
Thr at position 5 in the amino acid sequence of SEQ ID NO: 6 has been substituted
with another amino acid;
- (j) an antibody that comprises a heavy chain variable region comprising the CDR1 of
(a), CDR2 of (d), and CDR3 comprising the amino acid sequence of SEQ ID NO: 3;
- (k) an antibody that comprises a light chain variable region comprising the CDR1 of
(e), CDR2 of (h), and CDR3 of (i); and
- (l) an antibody that comprises the heavy chain variable region of (j) and the light
chain variable region of (k);
[5] an anti-IL-6 receptor antibody of any one of:
(a) an antibody that comprises a heavy chain variable region comprising CDR1 in which
Ser at position 1 in the amino acid sequence of SEQ ID NO: 1 has been substituted
with another amino acid, CDR2 in which Thr at position 9 and Ser at position 16 in
the amino acid sequence of SEQ ID NO: 2 have been substituted with other amino acids,
and CDR3 in which Ser at position 1 and Thr at position 5 in the amino acid sequence
of SEQ ID NO: 3 have been substituted with other amino acids;
(b) an antibody that comprises a light chain variable region comprising CDR1 in which
Arg at position I in the amino acid sequence of SEQ ID NO: 4 has been substituted
with another amino acid, CDR2 in which Thr at position 2 and Arg at position 4 in
the amino acid sequence of SEQ ID NO:5 have been substituted with other amino acids,
and CDR3 in which Gln at position 1 and Thr at position 5 in the amino acid sequence
of SEQ ID NO:6 have been substituted with other amino acids;
(c) an antibody that comprises a heavy chain variable region comprising the amino
acid sequence of SEQ ID NO: 22;
(d) an antibody that comprises a light chain variable region comprising the amino
acid sequence of SEQ ID NO: 23;
(e) an antibody that comprises the heavy chain variable region of (a) and the light
chain variable region of (b); and
(f) an antibody that comprises the heavy chain variable region of (c) and the light
chain variable region of (d);
[6] a human antibody constant region of any one of:
- (a) a human antibody constant region that comprises deletions of both Gly at position
329 (446 in the EU numbering system) and Lys at position 330 (447 in the EU numbering
system) in the amino acid sequence of SEQ ID NO: 19;
- (b) a human antibody constant region that comprises deletions of both Gly at position
325 (446 in the EU numbering system) and Lys at position 326 (447 in the EU numbering
system) in the amino acid sequence of SEQ ID NO: 20; and
- (c) a human antibody constant region that comprises deletions of both Gly at position
326 (446 in the EU numbering system) and Lys at position 327 (447 in the EU numbering
system) in the amino acid sequence of SEQ ID NO: 21;
[7] an IgG2 constant region in which the amino acids at positions 209 (330 in the
EU numbering system), 210 (331 in the EU numbering system), and 218 (339 in the EU
numbering system) in the amino acid sequence of SEQ ID NO: 20 have been substituted
with other amino acids;
[8] an IgG2 constant region in which the amino acid at position 276 (397 in the EU
numbering system) in the amino acid sequence of SEQ ID NO: 20 has been substituted
with another amino acid;
[9] an IgG2 constant region in which the amino acid at position 14 (131 in the EU
numbering system), 102 (219 in the EU numbering system), and/or 16 (133 in the EU
numbering system) in the amino acid sequence of SEQ ID NO: 20 has been substituted
with another amino acid;
[10] the IgG2 constant region of [9], wherein the amino acids at positions 20 (137
in the EU numbering system) and 21 (13 8 in the EU numbering system) in the amino
acid sequence of SEQ ID NO: 20 have been substituted with other amino acids;
[11] an IgG2 constant region in which His at position 147 (268 in the EU numbering
system), Arg at position234 (355 in the EU numbering system), and/or GIn at position
298 (419 in the EU numbering system) in the amino acid sequence of SEQ ID NO: 20 has
been substituted with another amino acid;
[12] an IgG2 constant region in which the amino acids at positions 209 (330 in the
EU numbering system), 210 (331 in the EU numbering system), 218 (339 in the EU numbering
system), 276 (397 in the EU numbering system), 14 (131 in the EU numbering system),
16 (133 in the EU numbering system), 102 (219 in the EU numbering system), 20 (137
in the EU numbering system), and 21 (138 in the EU numbering system) in the amino
acid sequence of SEQ ID NO: 20 have been substituted with other amino acids;
[13] the IgG2 constant region of [12], which further comprises deletions of both Gly
at position 325 (446 in the EU numbering system) and Lys at position 326 (447 in the
EU numbering system);
[14] an IgG2 constant region in which the amino acids at positions 276 (397 in the
EU numbering system), 14 (131 in the EU numbering system), 16 (133 in the EU numbering
system), 102 (219 in the EU numbering system), 20 (137 in the EU numbering system),
and 21 (138 in the EU numbering system) in the amino acid sequence of SEQ ID NO: 20
have been substituted with other amino acids;
[15] the IgG2 constant region of [14], which further comprises deletions of both Gly
at position 325 (446 in the EU numbering system) and Lys at position 326 (447 in the
EU numbering system);
[16] an IgG2 constant region in which the Cys at position 14 (131 in the EU numbering
system), Arg at position 16 (133 in the EU numbering system), Cys at position 102
(219 in the EU numbering system), Glu at position 20 (137 in the EU numbering system),
Ser at position 21 (138 in the EU numbering system), His at position 147 (268 in the
EU numbering system), Arg at position 234 (355 in the EU numbering system), and Gln
at position 298 (419 in the EU numbering system) in the amino acid sequence of SEQ
ID NO: 20 have been substituted with other amino acids;
[17] the IgG2 constant region of [16], which further comprises deletions of both Gly
at position 325 (446 in the EU numbering system) and Lys at position 326 (447 in the
EU numbering system);
[18] an IgG2 constant region in which the Cys at position 14 (131 in the EU numbering
system), Arg at position 16 (133 in the EU numbering system), Cys at position 102
(219 in the EU numbering system), Glu at position 20 (137 in the EU numbering system),
Ser at position 21 (138 in the EU numbering system), His at position 147 (268 in the
EU numbering system), Arg at position 234 (355 in the EU numbering system), Gln at
position 298 (419 in the EU numbering system), and Asn at position 313 (434 in the
EU numbering system) in the amino acid sequence of SEQ ID NO: 20 have been substituted
with other amino acids;
[19] the IgG2 constant region of [18], which further comprises deletions of both Gly
at position 325 (446 in the EU numbering system) and Lys at position 326 (447 in the
EU numbering system);
[20] an IgG4 constant region in which the amino acid at position 289 (409 in the EU
numbering system) in the amino acid sequence of SEQ ID NO: 21 has been substituted
with another amino acid;
[21] an IgG4 constant region in which the amino acids at position 289 (409 in the
EU numbering system), positions 14, 16, 20, 21, 97, 100, 102, 103, 104, and 105 (131,
133, 137, 138, 214, 217, 219, 220, 221, and 222 in the EU numbering system, respectively),
and positions 113, 114, and 115 (233, 234, and 235 in the EU numbering system, respectively),
have been substituted with other amino acids, and the amino acid at position 116 (236
in the EU numbering system) has been deleted from the amino acid sequence of SEQ ID
NO: 21;
[22] the IgG4 constant region of [21], which further comprises deletions of both Gly
at position 326 (446 in the EU numbering system) and Lys at position 327 (447 in the
EU numbering system);
[23] an IgG2 constant region in which Ala at position 209 (330 in the EU numbering
system), Pro at position 210 (331 in the EU numbering system), Thr at position 218
(339 in the EU numbering system), Cys at position 14 (131 in the EU numbering system),
Arg at position 16 (133 in the EU numbering system), Cys at position 102 (219 in the
EU numbering system), Glu at position 20 (137 in the EU numbering system), and Ser
at position 21 (138 in the EU numbering system) in the amino acid sequence of SEQ
ID NO: 20 have been substituted with other amino acids;
[24] the IgG2 constant region of [23], which further comprises deletions of both Gly
at position 325 (446 in the EU numbering system) and Lys at position 326 (447 in the
EU numbering system);
[25] an IgG2 constant region in which Cys at position 14 (131 in the EU numbering
system), Arg at position 16 (133 in the EU numbering system), Cys at position 102
(219 in the EU numbering system), Glu at position 20 (137 in the EU numbering system),
and Ser at position 21 (138 in the EU numbering system) in the amino acid sequence
of SEQ ID NO: 20 have been substituted with other amino acids;
[26] the IgG2 constant region of [25], which further comprises deletions of both Gly
at position 325 (446 in the EU numbering system) and Lys at position 326 (447 in the
EU numbering system);
[27] a constant region comprising the amino acid sequence of SEQ ID NO: 24;
[28] a constant region comprising the amino acid sequence of SEQ ID NO: 118;
[29] a constant region comprising the amino acid sequence of SEQ ID NO: 25;
[30] a constant region comprising the amino acid sequence of SEQ ID NO: 151;
[31] a constant region comprising the amino acid sequence of SEQ ID NO: 152;
[32] a constant region comprising the amino acid sequence of SEQ ID NO: 153;
[33] a constant region comprising the amino acid sequence of SEQ ID NO: 164;
[34] a human antibody constant region comprising the amino acid sequence of SEQ ID
NO: 194 (M40ΔGK);
[35] a human antibody constant region comprising the amino acid sequence of SEQ ID
NO: 192 (M86ΔGK);
[36] an antibody comprising the constant region of any one of [6] to [32];
[37] the antibody of [36], which binds to an IL-6 receptor;
[38] an anti-IL-6 receptor antibody whose binding activity to an IL-6 receptor is
1 nM or less;
[39] an anti-IL-6 receptor antibody, wherein the measured isoelectric point of the
full-length antibody is 7.0 or lower or the theoretical isoelectric point of the variable
region is 5.0 or lower;
[40] an anti-IL-6 receptor antibody, wherein the increase in the ratio of antibody
aggregate after one month at 25°C in a buffer containing 20 mM Histidine-HCl and 150
mM NaCl at pH 6.5 to 7.0 is 0.3% or less when the concentration of the antibody is
100 mg/ml; and
[41] a pharmaceutical composition comprising the antibody of any one of [36] to [40].
Brief Description of the Drawings
[0022]
Fig. 1 is a graph showing the BaF/gp130-neutralizing activities of WT and RD_6.
Fig. 2 is a graph showing a sensorgram for the interaction between rhIL-s6R (R&D systems)
and WT.
Fig. 3 is a graph showing a sensorgram for the interaction between rhIL-s6R (R&D systems)
and RD_6.
Fig. 4-1 is a diagram showing a list of CDR mutations that improve the affinity or
neutralizing activity in comparison with WT.
Fig. 4-2 is the continuation of Fig. 4-1.
Fig. 5 is a diagram showing a list of CDR mutations that in combination improve the
affinity or neutralizing activity.
Fig. 6 is a graph showing the BaF/gp130-neutralizing activities of WT and RDC23.
Fig. 7 is a graph showing a sensorgram for the interaction between rhIL-s6R (R&D systems)
and RDC23.
Fig. 8 is a graph showing a sensorgram for the interaction between rhsIL-6R and WT.
Fig. 9 is a graph showing a sensorgram for the interaction between rhsIL-6R and RDC23.
Fig. 10 is a graph showing a sensorgram for the interaction between SR344 and WT.
Fig. 11 is a graph showing a sensorgram for the interaction between SR344 and RDC23.
Fig. 12 is a graph showing the BaF/gp130-neutralizing activities of WT and H53L28.
Fig. 13 is a graph showing a sensorgram for the interaction between SR344 and H53/L28.
Fig. 14 is a graph showing transitions in the plasma concentrations of WT and H53/L28
after intravenous administration to mice.
Fig. 15 is a graph showing transitions in the plasma concentrations of WT and H53/L28
after subcutaneous administration to mice.
Fig. 16 is a graph showing the BaF/gp130-neutralizing activities of WT and PF1.
Fig. 17 is a graph showing a sensorgram for the interaction between SR344 and PF1.
Fig. 18 is a graph showing the result of testing the stability of WT and PF1 at high
concentrations.
Fig. 19 is a graph showing transitions in the plasma concentrations of WT and PF1
after intravenous administration to human IL-6 receptor transgenic mice.
Fig. 20 is a graph showing transitions in the plasma concentrations of free human
soluble IL-6 receptor after intravenous administration of WT or PF1 to human IL-6
receptor transgenic mice.
Fig. 21 is a graph showing the result of using gel filtration chromatography to analyze
the content of aggregates in WT-IgG1, WT-IgG2, WT-IgG4, IgG2-M397V, and IgG4-R409K
purified by hydrochloric acid elution.
Fig. 22 is a diagram showing the result of cation exchange chromatography (IEC) analysis
of WT-IgG1, WT-IgG2, and WT-IgG4.
Fig. 23 is a diagram showing predicted disulfide bonding in the hinge region of WT-IgG2.
Fig. 24 is a diagram showing predicted disulfide bonding in the hinge region of WT-IgG2-SKSC.
Fig. 25 is a diagram showing the result of cation exchange chromatography (IEC) analysis
of WT-IgG2 and IgG2-SKSC.
Fig. 26 is a diagram showing the result of cation exchange chromatography (IEC) analysis
of humanized PM-1 antibody, H chain C-terminal ΔK antibody, and H chain C-terminal
ΔGK antibody.
Fig. 27 shows comparison of the amounts WT-IgG1 WT-IgG2, WT-IgG4, WT-M14ΔGK, WT-M17ΔGK,
and WT-M11ΔGK bound to FcγRI.
Fig. 28 is a graph showing comparison of the amounts WT-IgG1, WT-IgG2, WT-IgG4, WT-M14ΔGK,
WT-M17ΔGK, and WT-M11ΔGK bound to FcγRIIa.
Fig. 29 is a graph showing comparison of the amounts WT-IgG1, WT-IgG2, WT-IgG4, WT-M14ΔGK,
WT-M17ΔGK, and WT-M11ΔGK bound to FcγRIIb.
Fig. 30 is a graph showing comparison of the amounts WT-IgG1, WT-IgG2, WT-IgG4, WT-M14ΔGK,
WT-M17ΔGK, and WT-M11ΔGK bound to FcγRIIIa (Val).
Fig. 31 is a graph showing the increase of aggregation in a stability test for WT-IgG1,
WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK at high concentrations.
Fig. 32 is a graph showing the increase of Fab fragments in a stability test for WT-IgG1,
WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK at high concentrations.
Fig. 33 is a diagram showing the result of cation exchange chromatography (IEC) analysis
of WT-IgG2, WT-M14ΔGK, and WT-M31ΔGK.
Fig. 34 is a graph showing the BaF/gp130-neutralizing activities of WT and F2H/L39-IgG1.
Fig. 35 is a graph showing the plasma concentration time courses of antibodies after
subcutaneous administration of WT, PF1, or F2H/L39-IgG1 at 1.0 mg/kg to cynomolgus
monkeys.
Fig. 36 is a graph showing the time courses of CRP concentration in the groups of
cynomolgus monkeys administered with WT or F2H/L39-IgG1.
Fig. 37 is a graph showing the time courses of free cynomolgus monkey IL-6 receptor
concentration in the groups of cynomolgus monkeys administered with WT or F2H/L39-IgG1.
Fig. 38 is a graph showing the time courses of plasma concentrations of WT-IgG1 and
WT-M14 after intravenous administration to human FcRn transgenic mice.
Fig. 39 is a graph showing the time courses of plasma concentrations of WT-IgG1, WT-M14,
and WT-M58 after intravenous administration to human FcRn transgenic mice.
Fig. 40 is a graph showing the time courses of plasma concentrations of WT-TgG1, WT-M44,
WT-M58, and WT-M73 after intravenous administration to human FcRn transgenic mice.
Fig. 41 is a diagram showing a cation exchange chromatography-based assessment of
the effect on heterogeneity by the constant region of anti IL-6 receptor antibodies
WT and F2H/L39, anti-IL-31 receptor antibody H0L0, and anti-RANKL antibody DNS.
Fig. 42 is a diagram showing a cation exchange chromatography-based assessment of
the effect on heterogeneity by the CH1 domain cysteine of anti IL-6 receptor antibodies
WT and F2H/L39.
Fig. 43 is a diagram showing a DSC-based assessment of the effect on denaturation
peak by the CHl domain cysteine of anti IL-6 receptor antibody WT.
Fig. 44 is a graph showing the activities of TOCILIZUMAB, the control, and Fv5-M83
to neutralize BaF/g130.
Fig. 45 is a graph showing the activities of TOCILIZUMAB, Fv3-M73, and Fv4-M73 to
neutralize BaF/gp130.
Fig. 46 is a graph showing the plasma concentration time courses of TOCILIZUMAB, the
control, Fv3-M73, Fv4-M73, and Fv5-M83 in cynomolgus monkeys after intravenous administration.
Fig. 47 is a graph showing the time courses of CRP concentration in cynomolgus monkeys
after intravenous administration of TOCILIZUMAB, the control, Fv3-M73, Fv4-M73, or
Fv5-M83.
Fig. 48 is a graph showing the time courses of percentage of soluble IL-6 receptor
neutralization in cynomolgus monkeys after intravenous administration of TOCILIZUMAB,
the control, Fv3-M73, Fv4-M73, or Fv5-M83.
Mode for Carrying Out the Invention
[0023] The present invention provides pharmaceutical compositions comprising second-generation
molecules that are more superior to the humanized anti-IL-6 receptor IgG1 antibody
TOCILIZUMAB, and have been improved to exhibit enhanced drug efficacy and pharmacokinetics,
and thus produce a prolonged therapeutic effect even when the frequency of administration
is reduced. They have also been improved to have reduced immunogenicity and improved
safety and physicochemical properties, by altering amino acid sequences of the variable
and constant regions of TOCILIZUMAB; and methods for producing such pharmaceutical
compositions. The present invention also provides antibody constant regions that are
suitable for pharmaceuticals.
[0024] The present invention relates to anti-IL-6 receptor antibodies exhibiting superior
antigen-binding activity, neutralizing activity, retention in plasma, stability, and/or
homogeneity, and reduced immunogenicity risk.
[0025] Preferably, the anti-IL-6 receptor antibody is a humanized PM-1 antibody (TOCILIZUMAB).
More specifically, the present invention provides humanized PM-1 antibodies with enhanced
antigen-binding activity, humanized PM-1 antibodies with enhanced neutralizing activity,
humanized PM-1 antibodies showing improved pharmacokinetics, humanized PM-1 antibodies
with reduced immunogenicity risk, humanized PM-1 antibodies with improved stability,
and humanized PM-1 antibodies with improved homogeneity, all of which have been achieved
through amino acid substitution.
[0026] Humanized PM-1 antibodies bind to the human IL-6 receptor, and thus inhibit the binding
between human IL-6 and the human IL-6 receptor. Herein, SEQ IDs in the Sequence Listing
correspond to the amino acid sequences of humanized PM-1 antibodies shown below.
Heavy chain amino acid sequence: SEQ ID NO: 15
Light chain amino acid sequence: SEQ ID NO: 16
Heavy chain variable region amino acid sequence: SEQ ID NO: 17
Light chain variable region amino acid sequence: SEQ ID NO: 18
Heavy chain CDR1 (HCDR1) amino acid sequence: SEQ ID NO: 1
Heavy chain CDR2 (HCDR2) amino acid sequence: SEQ ID NO: 2
Heavy chain CDR3 (HCDR3) amino acid sequence: SEQ ID NO: 3
Heavy chain FR1 (HFR1) amino acid sequence: SEQ ID NO: 7
Heavy chain FR2 (HFR2) amino acid sequence: SEQ ID NO: 8
Heavy chain FR3 (HFR3) amino acid sequence: SEQ ID NO: 9
Heavy chain FR4 (HFR4) amino acid sequence: SEQ ID NO: 10
Light chain CDR1 (LCDR1) amino acid sequence: SEQ ID NO: 4
Light chain CDR2 (LCDR2) amino acid sequence: SEQ ID NO: 5
Light chain CDR3 (LCDR3) amino acid sequence: SEQ ID NO: 6
Light chain FR1 (LFR1) amino acid sequence: SEQ ID NO: 11
Light chain FR2 (LFR2) amino acid sequence: SEQ ID NO: 12
Light chain FR3 (LFR3) amino acid sequence: SEQ ID NO: 13
Light chain FR4 (LFR4) amino acid sequence: SEQ ID NO: 14
<Antibodies with enhanced affinity and neutralizing activity>
[0027] The present invention provides anti-human IL-6 receptor antibodies exhibiting strong
human IL-6 receptor-binding and/or neutralizing activity. More specifically, the present
invention provides the following antibodies of (a) to (y), and methods for producing
the antibodies:
(a) An anti-human IL-6 receptor antibody comprising a heavy chain CDR1 in which Ser
at position 1 in the amino acid sequence of SEQ ID NO: 1 (HCDR1) has been substituted
with another amino acid.
[0028] The type of amino acid after substitution is not particularly limited; however, substitution
to Trp (RD_68), Thr (RD_37), Asp (RD_8), Asn (RD_11), Arg (RD_31), Val (RD_32), Phe
(RD_33), Ala (RD_34), Gln (RD_35), Tyr (RD_36), Leu (RD_38), His (RD_42), Glu (RD_45),
or Cys (RD_46) is preferred.
[0029] A sequence resulting from the substitution of Trp for Ser at position 1 in the amino
acid sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 26.
[0030] A sequence resulting from the substitution of Thr for Ser at position 1 in the amino
acid sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 27.
[0031] A sequence resulting from the substitution of Asp for Ser at position 1 in the amino
acid sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 28.
[0032] A sequence resulting from the substitution of Asn for Ser at position 1 in the amino
acid sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 29.
[0033] A sequence resulting from the substitution of Arg for Ser at position 1 in the amino
acid sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 30.
[0034] A sequence resulting from the substitution of Val for Ser at position 1 in the amino
acid sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 31.
[0035] A sequence resulting from the substitution of Phe for Ser at position 1 in the amino
acid sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 32.
[0036] A sequence resulting from the substitution of Ala for Ser at position 1 in the amino
acid sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 33.
[0037] A sequence resulting from the substitution of Gln for Ser at position 1 in the amino
acid sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 34.
[0038] A sequence resulting from the substitution of Tyr for Ser at position 1 in the amino
acid sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 35.
[0039] A sequence resulting from the substitution of Leu for Ser at position 1 in the amino
acid sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 36.
[0040] A sequence resulting from the substitution of His for Ser at position 1 in the amino
acid sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 37.
[0041] A sequence resulting from the substitution of Glu for Ser at position 1 in the amino
acid sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 38.
[0042] A sequence resulting from the substitution of Cys for Ser at position 1 in the amino
acid sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 39.
(b) An anti-human IL-6 receptor antibody comprising a heavy chain CDR1 in which Trp
at position 5 in the amino acid sequence of SEQ ID NO: 1 (HCDR1) has been substituted
with another amino acid.
[0043] The type of amino acid after substitution is not particularly limited; however, substitution
to Ile (RD_9) or Val (RD_30) is preferred.
[0044] A sequence resulting from the substitution of Ile for Trp at position 5 in the amino
acid sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 40.
[0045] A sequence resulting from the substitution of Val for Trp at position 5 in the amino
acid sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 41.
(c) An anti-human IL-6 receptor antibody comprising a heavy chain CDR2 in which Tyr
at position 1 in the amino acid sequence of SEQ ID NO: 2 (HCDR2) has been substituted
with another amino acid.
[0046] The type of amino acid after substitution is not particularly limited; however, substitution
to Phe (RD_82) is preferred.
[0047] A sequence resulting from the substitution of Phe for Tyr at position 1 in the amino
acid sequence of SEQ ID NO: 2 is shown in SEQ ID NO: 42.
(d) An anti-human IL-6 receptor antibody comprising a heavy chain CDR2 in which Thr
at position 8 in the amino acid sequence of SEQ ID NO: 2 (HCDR2) has been substituted
with another amino acid.
[0048] The type of amino acid after substitution is not particularly limited; however, substitution
to Arg (RD_79) is preferred.
[0049] A sequence resulting from the substitution of Arg for Thr at position 8 in the amino
acid sequence of SEQ ID NO: 2 is shown in SEQ ID NO: 43.
(e) An anti-human IL-6 receptor antibody comprising a heavy chain CDR2 in which Thr
at position 9 in the amino acid sequence of SEQ ID NO: 2 (HCDR2) has been substituted
with another amino acid.
[0050] The type of amino acid after substitution is not particularly limited; however, substitution
to Ser (RD_12) or Asn (RD_61) is preferred.
[0051] A sequence resulting from the substitution of Ser for Thr at position 9 in the amino
acid sequence of SEQ ID NO: 2 is shown in SEQ ID NO: 44.
[0052] A sequence resulting from the substitution of Asn for Thr at position 9 in the amino
acid sequence of SEQ ID NO: 2 is shown in SEQ ID NO: 45.
(f) An anti-human IL-6 receptor antibody comprising a heavy chain CDR3 in which Ser
at position 1 in the amino acid sequence of SEQ ID NO: 3 (HCDR3) has been substituted
with another amino acid.
[0053] The type of amino acid after substitution is not particularly limited; however, substitution
to Ile (RD_2), Val (RD_4), Thr (RD_80), or Leu (RD_5) is preferred.
[0054] A sequence resulting from the substitution of Ile for Ser at position 1 in the amino
acid sequence of SEQ ID NO: 3 is shown in SEQ ID NO: 46.
[0055] A sequence resulting from the substitution of Val for Ser at position 1 in the amino
acid sequence of SEQ ID NO: 3 is shown in SEQ ID NO: 47.
[0056] A sequence resulting from the substitution of Thr for Ser at position 1 in the amino
acid sequence of SEQ ID NO: 3 is shown in SEQ ID NO: 48.
[0057] A sequence resulting from the substitution of Leu for Ser at position 1 in the amino
acid sequence of SEQ ID NO: 3 is shown in SEQ ID NO: 49.
(g) An anti-human IL-6 receptor antibody comprising a heavy chain CDR3 in which Leu
at position 2 in the amino acid sequence of SEQ ID NO: 3 (HCDR3) has been substituted
with another amino acid.
[0058] The type of amino acid after substitution is not particularly limited; however, substitution
to Thr (RD_84) is preferred.
[0059] A sequence resulting from the substitution of Thr for Leu at position 2 in the amino
acid sequence of SEQ ID NO: 3 is shown in SEQ ID NO: 50.
(h) An anti-human IL-6 receptor antibody comprising a heavy chain CDR3 in which Thr
at position 5 in the amino acid sequence of SEQ ID NO: 3 (HCDR3) has been substituted
with another amino acid.
[0060] The type of amino acid after substitution is not particularly limited; however, substitution
to Ala (RD_3) or Ile (RD_83) is preferred. In addition, the substitution of Ser (RDC_14H)
for Thr at position 5 is also preferred.
[0061] A sequence resulting from the substitution of Ala for Thr at position 5 in the amino
acid sequence of SEQ ID NO: 3 is shown in SEQ ID NO: 51.
[0062] A sequence resulting from the substitution of Ile for Thr at position 5 in the amino
acid sequence of SEQ ID NO: 3 is shown in SEQ ID NO: 52.
[0063] A sequence resulting from the substitution of Ser for Thr at position 5 in the amino
acid sequence of SEQ ID NO: 3 is shown in SEQ ID NO: 53.
(i) An anti-human IL-6 receptor antibody comprising a heavy chain CDR3 in which Ala
at position 7 in the amino acid sequence of SEQ ID NO: 3 (HCDR3) has been substituted
with another amino acid.
[0064] The type of amino acid after substitution is not particularly limited; however, substitution
to Ser (RD_81) or Val (PF_3H) is preferred.
[0065] A sequence resulting from the substitution of Ser for Ala at position 7 in the amino
acid sequence of SEQ ID NO: 3 is shown in SEQ ID NO: 54.
[0066] A sequence resulting from the substitution of Val for Ala at position 7 in the amino
acid sequence of SEQ ID NO: 3 is shown in SEQ ID NO: 55.
(j) An anti-human IL-6 receptor antibody comprising a heavy chain CDR3 in which Met
at position 8 in the amino acid sequence of SEQ ID NO: 3 (HCDR3) has been substituted
with another amino acid.
[0067] The type of amino acid after substitution is not particularly limited; however, substitution
to Leu (PF_4H) is preferred.
[0068] A sequence resulting from the substitution of Leu for Met at position 8 in the amino
acid sequence of SEQ ID NO: 3 is shown in SEQ ID NO: 56.
(k) An anti-human IL-6 receptor antibody comprising a heavy chain CDR3 in which Ser
at position 1 and Thr at position 5 in the amino acid sequence of SEQ ID NO: 3 (HCDR3)
have been substituted with other amino acids.
[0069] The type of amino acid after substitution is not particularly limited; however, substitutions
of Leu for Ser at position 1 and Ala for Thr at position 5 (RD_6) are preferred. Other
preferred substitutions include: substitutions of Val for Ser at position 1 and Ala
for Thr at position 5 (RDC_2H); substitutions of Ile for Ser at position 1 and Ala
for Thr at position 5 (RDC_3H); substitutions of Thr for Ser at position 1 and Ala
for Thr at position 5 (RDC_4H); substitutions of Val for Ser at position 1 and Ile
for Thr at position 5 (RDC_5H); substitutions of Ile for Ser at position I and Ile
for Thr at position 5 (RDC_6H); substitutions of Thr for Ser at position 1 and Ile
for Thr at position 5 (RDC_7H); and substitutions of Leu for Ser at position 1 and
Ile for Thr at position 5 (RDC_8H).
[0070] A sequence resulting from the substitutions of Leu for Ser at position 1 and Ala
for Thr at position 5 in the amino acid sequence of SEQ ID NO: 3 is shown in SEQ ID
NO: 57.
[0071] A sequence resulting from the substitutions of Val for Ser at position 1 and Ala
for Thr at position 5 in the amino acid sequence of SEQ ID NO: 3 is shown in SEQ ID
NO: 58.
[0072] A sequence resulting from the substitutions of Ile for Ser at position 1 and Ala
for Thr at position 5 in the amino acid sequence of SEQ ID NO: 3 is shown in SEQ ID
NO: 59.
[0073] A sequence resulting from the substitutions of Thr for Ser at position 1 and Ala
for Thr at position 5 in the amino acid sequence of SEQ ID NO: 3 is shown in SEQ ID
NO: 60.
[0074] A sequence resulting from the substitutions of Val for Ser at position 1 and Ile
for Thr at position 5 in the amino acid sequence of SEQ ID NO: 3 is shown in SEQ ID
NO: 61.
[0075] A sequence resulting from the substitutions of Ile for Ser at position 1 and Ile
for Thr at position 5 in the amino acid sequence of SEQ ID NO: 3 is shown in SEQ ID
NO: 62.
[0076] A sequence resulting from the substitutions of Thr for Ser at position 1 and Ile
for Thr at position 5 in the amino acid sequence of SEQ ID NO: 3 is shown in SEQ ID
NO: 63.
[0077] A sequence resulting from the substitutions of Leu for Ser at position 1 and Ile
for Thr at position 5 in the amino acid sequence of SEQ ID NO: 3 is shown in SEQ ID
NO: 64.
(l) An anti-human IL-6 receptor antibody comprising a heavy chain CDR3 in which Leu
at position 2, Ala at position 7, and Met at position 8 in the amino acid sequence
of SEQ ID NO: 3 (HCDR3) have been substituted with other amino acids.
[0078] The type of amino acid after substitution is not particularly limited; however, substitutions
of Thr for Leu at position 2, Val for Ala at position 7, and Leu for Met at position
8 (RD_78) are preferred.
[0079] A sequence resulting from the substitutions of Thr for Leu at position 2, Val for
Ala at position 7, and Leu for Met at position 8 in the amino acid sequence of SEQ
ID NO: 3 is shown in SEQ ID NO: 65.
(m) An anti-human IL-6 receptor antibody comprising a light chain CDR1 in which Arg
at position 1 in the amino acid sequence of SEQ ID NO: 4 (LCDR1) has been substituted
with another amino acid.
[0080] The type of amino acid after substitution is not particularly limited; however, substitution
to Phe (RD_18) is preferred.
[0081] A sequence resulting from the substitution of Phe for Arg at position 1 in the amino
acid sequence of SEQ ID NO: 4 is shown in SEQ ID NO: 66.
(n) An anti-human IL-6 receptor antibody comprising a light chain CDR1 in which Gln
at position 4 in the amino acid sequence of SEQ ID NO: 4 (LCDR1) has been substituted
with another amino acid.
[0082] The type of amino acid after substitution is not particularly limited; however, substitution
to Arg (RD_26) or Thr (RD_20) is preferred.
[0083] A sequence resulting from the substitution of Arg for Gln at position 4 in the amino
acid sequence of SEQ ID NO: 4 is shown in SEQ ID NO: 67.
[0084] A sequence resulting from the substitution of Thr for Gln at position 4 in the amino
acid sequence of SEQ ID NO: 4 is shown in SEQ ID NO: 68.
(o) An anti-human IL-6 receptor antibody comprising a light chain CDR1 in which Tyr
at position 9 in the amino acid sequence of SEQ ID NO: 4 (LCDR1) has been substituted
with another amino acid.
[0085] The type of amino acid after substitution is not particularly limited; however, substitution
to Phe (RD_73) is preferred.
[0086] A sequence resulting from the substitution of Phe for Tyr at position 9 in the amino
acid sequence of SEQ ID NO: 4 is shown in SEQ ID NO: 69.
(p) An anti-human IL-6 receptor antibody comprising a light chain CDR1 in which Asn
at position 11 in the amino acid sequence of SEQ ID NO: 4 (LCDR1) has been substituted
with another amino acid.
[0087] The type of amino acid after substitution is not particularly limited; however, substitution
to Ser (RD_27) is preferred.
[0088] A sequence resulting from the substitution of Ser for Asn at position 11 in the amino
acid sequence of SEQ ID NO: 4 is shown in SEQ ID NO: 70.
(q) An anti-human IL-6 receptor antibody comprising a light chain CDR2 in which Thr
at position 2 in the amino acid sequence of SEQ ID NO: 5 (LCDR2) has been substituted
with another amino acid.
[0089] The type of amino acid after substitution is not particularly limited; however, substitution
to Gly is preferred.
[0090] A sequence resulting from the substitution of Gly for Thr at position 2 in the amino
acid sequence of SEQ ID NO: 5 is shown in SEQ ID NO: 71.
(r) An anti-human IL-6 receptor antibody comprising a light chain CDR3 in which Gln
at position 1 in the amino acid sequence of SEQ ID NO: 6 (LCDR3) has been substituted
with another amino acid.
[0091] The type of amino acid after substitution is not particularly limited; however substitution
to Gly (RD_28), Asn (RD_29), or Ser (RDC_15L) is preferred.
[0092] A sequence resulting from the substitution of Gly for Gln at position 1 in the amino
acid sequence of SEQ ID NO: 6 is shown in SEQ ID NO: 72.
[0093] A sequence resulting from the substitution of Asn for Gln at position 1 in the amino
acid sequence of SEQ ID NO: 6 is shown in SEQ ID NO: 73.
[0094] A sequence resulting from the substitution of Ser for Gln at position I in the amino
acid sequence of SEQ ID NO: 6 is shown in SEQ ID NO: 74.
(s) An anti-human IL-6 receptor antibody comprising a light chain CDR3 in which Gly
at position 3 in the amino acid sequence of SEQ ID NO: 6 has been substituted with
another amino acid.
[0095] The type of amino acid after substitution is not particularly limited; however, substitution
to Ser is preferred.
[0096] A sequence resulting from the substitution of Ser for Gly at position 3 in the amino
acid sequence of SEQ ID NO: 6 is shown in SEQ ID NO: 75.
(t) An anti-human IL-6 receptor antibody comprising a light chain CDR1 in which Tyr
at position 9 in the amino acid sequence of SEQ ID NO: 4 (LCDR1) has been substituted
with another amino acid, and a light chain CDR3 in which Gly at position 3 in the
amino acid sequence of SEQ ID NO: 6 (LCDR3) has been substituted with another amino
acid.
[0097] The type of amino acid after substitution is not particularly limited; however, Tyr
at position 9 in the amino acid sequence of SEQ ID NO: 4 (LCDR1) is preferably substituted
with Phe, while Gly at position 3 in the amino acid sequence of SEQ ID NO: 6 (LCDR3)
is preferably substituted with Ser (RD_72).
(u) An anti-human IL-6 receptor antibody comprising a light chain CDR3 in which Thr
at position 5 in the amino acid sequence of SEQ ID NO: 6 (LCDR3) has been substituted
with another amino acid.
[0098] The type of amino acid after substitution is not particularly limited; however, substitution
to Arg (RD_23) or Ser is preferred.
[0099] A sequence resulting from the substitution of Arg for Thr at position 5 in the amino
acid sequence of SEQ ID NO: 6 is shown in SEQ ID NO: 76.
[0100] A sequence resulting from the substitution of Ser for Thr at position 5 in the amino
acid sequence of SEQ ID NO: 6 is shown in SEQ ID NO: 77.
(v) An anti-human IL-6 receptor antibody comprising a light chain CDR3 in which Gln
at position 1 and Thr at position 5 in the amino acid sequence of SEQ ID NO: 6 (LCDR3)
have been substituted with other amino acids.
[0101] The type of amino acid after substitution is not particularly limited; however, substitutions
of Gly for Gln at position 1 and Ser for Thr at position 5 (RD_22) are preferred.
Other preferred substitutions include substitutions of Gly for Gln at position 1 and
Arg for Thr at position 5 (RDC_11L).
[0102] A sequence resulting from the substitutions of Gly for Gln at position 1 and Ser
for Thr at position 5 in the amino acid sequence of SEQ ID NO: 6 is shown in SEQ ID
NO: 78.
[0103] A sequence resulting from the substitutions of Gly for Gln at position 1 and Arg
for Thr at position 5 in the amino acid sequence of SEQ ID NO: 6 is shown in SEQ ID
NO: 79.
[0104] (w) An anti-human IL-6 receptor antibody comprising a heavy chain CDR2 in which Thr
at position 9 in the amino acid sequence of SEQ ID NO: 2 (HCDR2) has been substituted
with another amino acid, and a heavy chain CDR3 in which Ser at position 1 and Thr
at position 5 in the amino acid sequence of SEQ ID NO: 3 (HCDR3) have been substituted
with other amino acids.
[0105] Thr at position 9 in the amino acid sequence of SEQ ID NO: 2 (HCDR2) is preferably
replaced with Asn. Furthermore, preferred combinations of amino acids for substitutions
of Ser at position 1 and Thr at position 5 in the amino acid sequence of SEQ ID NO:
3 (HCDR3) include: Leu and Ala (RDC_27H); Val and Ala (RDC_28H); Ile and Ala (RDC_30H);
Thr and Ala (RDC_4H); Val and Ile (RDC_29H); Ile and Ile (RDC_32H); Thr and Ile (RDC_7H);
and Leu and Ile (RDC_8H).
(x) An antibody that comprises a variable region comprising the heavy chain CDR3 of
(k) and a variable region comprising the light chain CDR3 of (v).
(y) The antibody of (x), which further comprises the heavy chain CDR2 of (e).
[0106] The present invention provides antibodies comprising at least the amino acid substitution
of any one of (a) to (y) described above and methods for producing the antibodies.
Thus, the antibodies of the present invention can also comprise other amino acid substitutions
in addition to the amino acid substitution of any one of (a) to (y) described above.
Furthermore, the antibodies of the present invention also include antibodies comprising
a combination of any amino acid substitutions of (a) to (y) described above. The amino
acid substitutions of (a) to (y) described above include substitutions of the CDR
amino acid sequences described above to other amino acids. Amino acid substitutions
other than those of (a) to (y) described above include, for example, amino acid sequence
substitutions, deletions, additions, and/or insertions in other CDR regions. Such
substitutions also include amino acid sequence substitutions, deletions, additions,
and/or insertions in the FR regions. Such substitutions further include substitutions,
deletions, additions, and/or insertions in the constant regions.
[0107] Furthermore, the antibodies of the present invention also include antibodies in which
a high affinity CDR discovered in the present invention is grafted into any framework
other than a humanized PM-1 antibody. The antibodies of the present invention also
include antibodies in which the loss of affinity as a result of grafting a high affinity
CDR discovered in the present invention into any framework other than a humanized
PM-1 antibody has been compensated by mutations introduced into the framework region
to restore the original affinity (see, for example,
Curr. Opin. Biotechnol. 1994 Aug;5(4):428-33), and antibodies in which the loss has been compensated by mutations introduced into
the CDR region to restore the original affinity (see, for example,
US 2006/0122377).
[0108] In the present invention, the amino acid substitution of any one of (a) to (y) described
above is preferably introduced into a humanized PM-1 antibody. Humanized PM-1 antibodies
introduced with the amino acid substitution of any one of (a) to (y) described above
have strong IL-6 receptor-neutralizing activity. Humanized PM-1 antibodies introduced
with the amino acid substitution of any one of (a) to (y) described above are effective
as therapeutic agents for IL-6-associated inflammatory diseases such as rheumatoid
arthritis.
[0109] Antibodies comprising the amino acid substitution of any one of (a) to (y) described
above can also be referred to as, for example, (1) or (2) described below. An example
of antibody comprising the substitution of (a) is described here; other antibodies
comprising the substitution of any one of (b) to (y) can also be referred to in the
same way.
- (1) An antibody that comprises a heavy chain variable region comprising CDR1 comprising
an amino acid sequence in which Ser at position 1 in the amino acid sequence of SEQ
ID NO: 1 has been substituted with another amino acid
- (2) An antibody that comprises an H chain comprising CDR1 comprising an amino acid
sequence in which Ser at position 1 in the amino acid sequence of SEQ ID NO: 1 has
been substituted with another amino acid
<Antibodies with enhanced binding activity>
[0110] The present invention further provides anti-IL-6 receptor antibodies with strong
IL-6 receptor-binding activity. Herein, "anti-IL-6 receptor antibodies with strong
IL-6 receptor-binding activity" typically refers to antibodies whose affinity is measured
to be 1 nM or less at 37°C under physiological conditions, preferably 0.1 nM or less,
and more preferably 0.04 nM or less. Such anti-IL-6 receptor antibodies with strong
IL-6 receptor binding activity are assumed to have an enhanced activity of neutralizing
the biological activity of the antigen.
[0111] There is no limitation on the type of amino acid substitutions introduced to the
present invention's anti-IL-6 receptor antibodies with strong IL-6 receptor binding
activity. Such amino acid substitutions include, for example, the above-described
amino acid substitutions.
[0112] The type of IL-6 receptor is not particularly limited; however, human IL-6 receptor
is preferred.
[0113] The binding activity can be determined by methods known to those skilled in the art,
for example, using Biacore (BIACORE) or such, based on surface plasmon resonance (SPR).
<Antibodies having a CDR sequence with reduced immunogenicity risk>
[0114] The present invention also provides anti-IL-6 receptor antibodies with reduced immunogenicity,
in particular, humanized PM-1 antibodies. The immunogenicity is assumed to be enhanced
when the sequence of an antibody contains a T-cell epitope that binds to HLA. Thus,
the immunogenicity risk for an antibody can be reduced by removing the T-cell epitope
from the antibody sequence through sequence substitution.
[0115] The present invention provides light chain variable regions of humanized anti-human
IL-6 receptor antibodies with reduced immunogenicity, in particular, those of humanized
PM-1 antibodies, from which T-cell epitopes have been removed through substituting
other amino acids in the antibody amino acid sequences, in particular, CDR sequences.
The present invention also provides antibodies comprising such light chain variable
regions.
[0116] More specifically, the present invention provides light chain CDR2 in which Thr at
position 2 in the amino acid sequence of SEQ ID NO: 5 (LCDR2) has been substituted
with another amino acid. The present invention also provides light chain variable
regions comprising such light chain CDR2. The present invention also provides anti-IL-6
receptor antibodies comprising such light chain variable region. The amino acid sequence
after substitution is not particularly limited; however, substitution to Gly is preferred.
A sequence comprising the substitution of Gly for Thr at position 2 in the amino acid
sequence of SEQ ID NO: 5 is shown in SEQ ID NO: 71. The amino acid substitution is
preferably introduced into a light chain variable region of a humanized PM-1 antibody.
<FR and CDR of H53/L28>
[0117] The present invention also provides anti-human IL-6 receptor antibodies with improved
pharmacokinetic, increased stability, and/or reduced immunogenicity. The half-lives
of IgGs sharing the same Fc domain in plasma have been found to be correlated to isoelectric
points with a high correlation coefficient. Then, the present inventors tried altering
the isoelectric points of the variable regions of two antibodies against different
antigens, and successfully controlled their half-lives in plasma without altering
their Fc domains irrespective of the antigen type. The rate of non-specific antibody
uptake by endothelial cells is assumed to depend on the physicochemical Coulomb interaction
between IgG and negatively charged cell surface. Lowering the isoelectric point of
IgG impairs the Coulomb interaction, which reduces the non-specific uptake by endothelial
cells, and as a result, the metabolism in endothelial cells is reduced. This can improve
pharmacokinetics.
[0118] Specifically, the present invention provides anti-human IL-6 receptor antibodies
with reduced isoelectric point and improved pharmacokinetics, by substituting amino
acids in the amino acid sequence of an anti-IL-6 receptor antibody, in particular,
a humanized PM-1 antibody. Specifically, the humanized PM-1 antibody is altered to
reduce its isoelectric point by substituting other amino acids at H13 (amino acid
at position 13 in SEQ ID NO: 7), H16 (amino acid at position 16 in SEQ ID NO: 7),
H43 (amino acid at position 8 in SEQ ID NO: 8), H81 (amino acid at position 16 in
SEQ ID NO: 9), H105 (amino acid at position 3 in SEQ ID NO: 10), L18 (amino acid at
position 18 in SEQ ID NO: 11), L45 (amino acid at position 11 in SEQ ID NO: 12), L79
(amino acid at position 23 in SEQ ID NO: 13), L107 (amino acid at position 10 in SEQ
ID NO: 14), H31 (amino acid at position 1 in SEQ ID NO: 1), L24 (amino acid at position
1 in SEQ ID NO: 4), and/or L53 (amino acid at position 4 in SEQ ID NO: 5), where positions
are numbered according to Kabat's numbering system (Kabat EA
et al., 1991 Sequences of Proteins of Immunological Interest. NIH). These substitutions can
lower the isoelectric point of a humanized PM-1 antibody without affecting its binding
activity and stability. Some amino acid residues originated from the mouse sequence
remain unsubstituted in the humanized PM-1 antibody to maintain its binding activity
even after humanization of the mouse sequence. More specifically, amino acids at H27
(amino acid at position 27 in SEQ ID NO: 7), H28 (amino acid at position 28 in SEQ
ID NO: 7), H29 (amino acid at position 29 in SEQ ID NO: 7), H30 (amino acid at position
30 in SEQ ID NO: 7), and H71 in the humanized PM-1 antibody (positions are numbered
according to Kabat's numbering system described above) are of the mouse sequence.
HFR1 can be converted into a human sequence by substituting H13, H16, H23, and H30,
which enables to produce an antibody whose immunogenicity risk is lower than that
of the humanized PM-1 antibody. Furthermore, since the humanized PM-1 antibody is
an antibody humanized by CDR grafting, its stability may be further improved. Antibodies
can be stabilized, for example, by substituting hydrophilic amino acids for amino
acid residues exposed on the surface of the antibody variable region. Alternatively,
antibodies can also be stabilized by altering the CDR sequence to a consensus sequence.
The humanized PM-1 antibody can be stabilized by a substitution of Ile for Met at
H69 (amino acid position 4 in SEQ ID NO: 9) (stabilization of the hydrophobic core),
Ser for Leu at H70 (amino acid at position 5 in SEQ ID NO: 9) (conversion of the surface-exposed
residue to a hydrophilic residue), Asn for Thr at H58 (amino acid at position 9 in
SEQ ID NO: 2) (alternation of the heavy chain CDR2 to a consensus sequence), Gly for
Ser at H65 (amino acid at position 16 in SEQ ID NO: 2) (substitution of Gly in the
β turn region and alternation of the heavy chain CDR2 to a consensus sequence), or
Ser for Thr at L93 (amino acid at position 5 in SEQ ID NO: 6) (conversion of the surface-exposed
residue to a hydrophilic residue) (positions are numbered according to Kabat's numbering
system described above). Alternatively, in
silico-predicted T-cell epitopes can be removed by substituting Gly for Thr at L51 at position
2 in LCDR2 (SEQ ID NO: 5) described above, and this can reduce the immunogenicity
risk without affecting the binding activity and stability, Anti-IL-6 receptor antibodies
with improved stability and antibody pharmacokinetics, as well as reduced immunogenicity
can be obtained by using these amino acid substitutions in combination.
[0119] Such antibodies include, for example, the antibodies of (1) to (37) below.
(1) An antibody that comprises a heavy chain variable region comprising FR1 in which
Arg at position 13 in the amino acid sequence of SEQ ID NO: 7 has been substituted
with another amino acid.
[0120] The type of amino acid after substitution is not particularly limited; however, substitution
to Lys is preferred.
[0121] A sequence resulting from the substitution of Lys for Arg at position 13 in the amino
acid sequence of SEQ ID NO: 7 is shown in SEQ ID NO: 80.
(2) An antibody that comprises a heavy chain variable region comprising FR1 in which
Gln at position 16 in the amino acid sequence of SEQ ID NO: 7 has been substituted
with another amino acid.
[0122] The type of amino acid after substitution is not particularly limited; however, substitution
of Glu is preferred.
[0123] A sequence resulting from the substitution of Glu for Gln at position 16 in the amino
acid sequence of SEQ ID NO: 7 is shown in SEQ ID NO: 81.
(3) An antibody that comprises a heavy chain variable region comprising FR1 in which
Thr at position 23 in the amino acid sequence of SEQ ID NO: 7 has been substituted
with another amino acid.
[0124] The type of amino acid after substitution is not particularly limited; however, substitution
to Ala is preferred.
[0125] A sequence resulting from the substitution of Ala for Thr at position 23 in the amino
acid sequence of SEQ ID NO: 7 is shown in SEQ ID NO: 82.
(4) An antibody that comprises a heavy chain variable region comprising FR1 in which
Thr at position 30 in the amino acid sequence of SEQ ID NO: 7 has been substituted
with another amino acid.
[0126] The type of amino acid after substitution is not particularly limited; however, substitution
to Ser is preferred.
[0127] A sequence resulting from the substitution of Ser for Thr at position 30 in the amino
acid sequence of SEQ ID NO: 7 is shown in SEQ ID NO: 83.
(5) An antibody that comprises a heavy chain variable region comprising FR1 in which
Arg at position 13, Gln at position 16, Thr at position 23, and Thr at position 30
in the amino acid sequence of SEQ ID NO: 7 have been substituted with other amino
acids.
[0128] The type of amino acid after substitution is not particularly limited; however, substitutions
of Lys for Arg at position 13, Glu for Gln at position 16, Ala for Thr at position
23, and Ser for Thr at position 30 are preferred.
[0129] A sequence resulting from the substitutions of Lys for Arg at position 13, Glu for
Gln at position 16, Ala for Thr at position 23, and Ser for Thr at position 30 in
the amino acid sequence of SEQ ID NO: 7 is shown in SEQ ID NO: 84.
(6) An antibody that comprises a heavy chain variable region comprising FR2 in which
Arg at position 8 in the amino acid sequence of SEQ ID NO: 8 has been substituted
with another amino acid.
[0130] The type of amino acid after substitution is not particularly limited; however, substitution
to Glu is preferred.
[0131] A sequence resulting from the substitution of Glu for Arg at position 8 in the amino
acid sequence of SEQ ID NO: 8 is shown in SEQ ID NO: 85.
(7) An antibody that comprises a heavy chain variable region comprising FR3 in which
Met at position 4 in the amino acid sequence of SEQ ID NO: 9 has been substituted
with another amino acid.
[0132] The type of amino acid after substitution is not particularly limited; however, substitution
to Ile is preferred.
[0133] A sequence resulting from the substitution of Ile for Met at position 4 in the amino
acid sequence of SEQ ID NO: 9 is shown in SEQ ID NO: 86.
(8) An antibody that comprises a heavy chain variable region comprising FR3 in which
Leu at position 5 in the amino acid sequence of SEQ ID NO: 9 has been substituted
with another amino acid.
[0134] The type of amino acid after substitution is not particularly limited; however, substitution
to Ser is preferred.
[0135] A sequence resulting from the substitution of Ser for Leu at position 5 in the amino
acid sequence of SEQ ID NO: 9 is shown in SEQ ID NO: 87.
(9) An antibody that comprises a heavy chain variable region comprising FR3 in which
Arg at position 16 in the amino acid sequence of SEQ ID NO: 9 has been substituted
with another amino acid.
[0136] The type of amino acid after substitution is not particularly limited; however, substitution
to Lys is preferred.
[0137] A sequence resulting from the substitution of Lys for Arg at position 16 in the amino
acid sequence of SEQ ID NO: 9 is shown in SEQ ID NO: 88.
(10) An antibody that comprises a heavy chain variable region comprising FR3 in which
Val at position 27 in the amino acid sequence of SEQ ID NO: 9 has been substituted
with another amino acid.
[0138] The type of amino acid after substitution is not particularly limited; however, substitution
to Ala is preferred.
[0139] A sequence resulting from the substitution of Ala for Val at position 27 in the amino
acid sequence of SEQ ID NO: 9 is shown in SEQ ID NO: 89.
(11) An antibody that comprises a heavy chain variable region comprising FR3 in which
Met at position 4, Leu at position 5, Arg at position 16, and Val at position 27 in
the amino acid sequence of SEQ ID NO: 9 (HFR3) have been substituted with other amino
acids.
[0140] The type of amino acid after substitution is not particularly limited; however, substitutions
of Ile for Met at position 4, Ser for Leu at position 5, Lys for Arg at position 16,
and Ala for Val at position 27 are preferred.
[0141] A sequence resulting from the substitutions of Ile for Met at position 4, Ser for
Leu at position 5, Lys for Arg at position 16, and Ala for Val at position 27 in the
amino acid sequence of SEQ ID NO: 9 is shown in SEQ ID NO: 90.
(12) An antibody that comprises a heavy chain variable region comprising FR4 in which
Gln at position 3 in the amino acid sequence of SEQ ID NO: 10 (HFR4) has been substituted
with another amino acid.
[0142] The type of amino acid after substitution is not particularly limited; however, substitution
to Glu is preferred.
[0143] A sequence resulting from the substitution of Glu for Gln at position 3 in the amino
acid sequence of SEQ ID NO: 10 is shown in SEQ ID NO: 91.
(13) An antibody that comprises a light chain variable region comprising FR1 in which
Arg at position 18 in the amino acid sequence of SEQ ID NO: 11 (LFR1) has been substituted
with another amino acid.
[0144] The type of amino acid after substitution is not particularly limited; however, substitution
to Ser is preferred.
[0145] A sequence resulting from the substitution of Ser for Arg at position 18 in the amino
acid sequence of SEQ ID NO: 11 is shown in SEQ ID NO: 92.
(14) An antibody that comprises a light chain variable region comprising FR2 in which
Lys at position 11 in the amino acid sequence of SEQ ID NO: 12 (LFR2) has been substituted
with another amino acid.
[0146] The type of amino acid after substitution is not particularly limited; however, substitution
to Glu is preferred.
[0147] A sequence resulting from the substitution of Glu for Lys at position 11 in the amino
acid sequence of SEQ ID NO: 12 is shown in SEQ ID NO: 93.
(15) An antibody that comprises a light chain variable region comprising FR3 in which
Gln at position 23 in the amino acid sequence of SEQ ID NO: 13 has been substituted
with another amino acid.
[0148] The type of amino acid after substitution is not particularly limited; however, substitution
to Glu is preferred.
[0149] A sequence resulting from the substitution of Glu for Gln at position 23 in the amino
acid sequence of SEQ ID NO: 13 is shown in SEQ ID NO: 94.
(16) An antibody that comprises a light chain variable region comprising FR3 in which
Pro at position 24 in the amino acid sequence of SEQ ID NO: 13 has been substituted
with another amino acid.
[0150] The type of amino acid after substitution is not particularly limited; however, substitution
to Ala is preferred.
[0151] A sequence resulting from the substitution of Ala for Pro at position 24 in the amino
acid sequence of SEQ ID NO: 13 is shown in SEQ ID NO: 95.
(17) An antibody that comprises a light chain variable region comprising FR3 in which
Ile at position 27 in the amino acid sequence of SEQ ID NO: 13 has been substituted
with another amino acid.
[0152] The type of amino acid after substitution is not particularly limited; however, substitution
to Ala is preferred.
[0153] A sequence resulting from the substitution of Ala for Ile at position 27 in the amino
acid sequence of SEQ ID NO: 13 is shown in SEQ ID NO: 96.
(18) An antibody that comprises a light chain variable region comprising FR3 in which
Gln at position 23, Pro at position 24, and Ile at position 27 in the amino acid sequence
of SEQ ID NO: 13 (LFR3) have been substituted with other amino acids.
[0154] The type of amino acid after substitution is not particularly limited; however, substitutions
of Glu for Gln at position 23, Ala for Pro at position 24, and Ala for Ile at position
27 are preferred.
[0155] A sequence resulting from the substitutions of Glu for Gln at position 23, Ala for
Pro at position 24, and Ala for Ile at position 27 in the amino acid sequence of SEQ
ID NO: 13 is shown in SEQ ID NO: 97.
(19) An antibody that comprises a light chain variable region comprising FR4 in which
Lys at position 10 in the amino acid sequence of SEQ ID NO: 14 (LFR4) has been substituted
with another amino acid.
[0156] The type of amino acid after substitution is not particularly limited; however, substitution
to Glu is preferred.
[0157] A sequence resulting from the substitution of Glu for Lys at position 10 in the amino
acid sequence of SEQ ID NO: 14 is shown in SEQ ID NO: 98.
(20) An antibody that comprises a heavy chain variable region comprising FR4 in which
Ser at position 5 in the amino acid sequence of SEQ ID NO: 10 (HFR4) has been substituted
with another amino acid.
[0158] The type of amino acid after substitution is not particularly limited; however, substitution
to Thr is preferred.
[0159] A sequence resulting from the substitution of Thr for Ser at position 5 in the amino
acid sequence of SEQ ID NO: 10 is shown in SEQ ID NO: 132.
(21) An antibody that comprises a heavy chain variable region comprising FR4 in which
Gln at position 3 and Ser at position 5 in the amino acid sequence of SEQ ID NO: 10
(HFR4) have been substituted with other amino acids.
[0160] The type of amino acid after substitution is not particularly limited; however, substitutions
of Glu for Gln at position 3 and Thr for Ser at position 5 are preferred.
[0161] A sequence resulting from the substitutions of Glu for Gln at position 3 and Thr
for Ser at position 5 in the amino acid sequence of SEQ ID NO: 10 is shown in SEQ
ID NO: 133.
(22) An antibody that comprises a heavy chain variable region of a humanized PM-1
antibody comprising the amino acid substitutions of (5), (6), (11), and (21).
(23) An antibody that comprises a light chain variable region of a humanized PM-1
antibody comprising the amino acid substitutions of (13), (14), (18), and (19).
(24) An antibody that comprises the heavy chain variable region of (22) and the light
chain variable region of (23).
(25) An antibody that comprises a heavy chain variable region comprising CDR1 in which
Ser at position 1 in the amino acid sequence of SEQ ID NO: 1 (HCDR1) has been substituted
with another amino acid.
[0163] The type of amino acid after substitution is not particularly limited; however, substitution
of Asp is preferred.
[0164] A sequence resulting from the substitution of Asp for Ser at position 1 in the amino
acid sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 28.
(26) An antibody that comprises a heavy chain variable region comprising CDR2 in which
Ser at position 16 in the amino acid sequence of SEQ ID NO: 2 has been substituted
with another amino acid.
[0165] The type of amino acid after substitution is not particularly limited; however, substitution
of Gly is preferred.
[0166] A sequence resulting from the substitution of Gly for Ser at position 16 in the amino
acid sequence of SEQ ID NO: 2 is shown in SEQ ID NO: 99.
(27) An antibody that comprises a heavy chain variable region comprising CDR2 in which
Thr at position 9 and Ser at position 16 in the amino acid sequence of SEQ ID NO:
2 (HCDR2) have been substituted with other amino acids.
[0167] The type of amino acid after substitution is not particularly limited; however, substitutions
of Asn for Thr at position 9 and Gly for Ser at position 16 are preferred.
[0168] A sequence resulting from the substitutions of Asn for Thr at position 9 and Gly
for Ser at position 16 in the amino acid sequence of SEQ ID NO: 2 is shown in SEQ
ID NO: 100.
(28) An antibody that comprises a light chain variable region comprising CDR1 in which
Arg at position 1 in the amino acid sequence of SEQ ID NO: 4 (LCDR1) has been substituted
with another amino acid.
[0169] The type of amino acid after substitution is not particularly limited; however, substitution
of Gln is preferred.
[0170] A sequence resulting from the substitution of Gln for Arg at position 1 in the amino
acid sequence of SEQ ID NO: 4 is shown in SEQ ID NO: 101.
(29) An antibody that comprises a light chain variable region comprising CDR2 in which
Arg at position 4 in the amino acid sequence of SEQ ID NO: 5 has been substituted
with another amino acid.
[0171] The type of amino acid after substitution is not particularly limited; however, substitution
to Glu is preferred.
[0172] A sequence resulting from the substitution of Glu for Arg at position 4 in the amino
acid sequence of SEQ ID NO: 5 is shown in SEQ ID NO: 102.
(30) An antibody that comprises a light chain variable region comprising CDR2 in which
Thr at position 2 and Arg at position 4 in the amino acid sequence of SEQ ID NO: 5
(LCDR2) have been substituted with other amino acids.
[0173] The type of amino acid after substitution is not particularly limited; however, substitutions
of Gly for Thr at position 2 and Glu for Arg at position 4 are preferred.
[0174] A sequence resulting from the substitutions of Gly for Thr at position 2 and Glu
for Arg at position 4 in the amino acid sequence of SEQ ID NO: 5 is shown in SEQ ID
NO: 103.
(31) An antibody that comprises a light chain variable region comprising CDR3 in which
Thr at position 5 in the amino acid sequence of SEQ ID NO: 6 (LCDR3) has been substituted
with another amino acid.
[0175] The type of amino acid after substitution is not particularly limited; however, substitution
to Ser is preferred.
[0176] A sequence resulting from the substitution of Ser for Thr at position 5 in the amino
acid sequence of SEQ ID NO: 6 is shown in SEQ ID NO: 77.
(32) An antibody that comprises a heavy chain variable region comprising the amino
acid substitutions of (25) and (27).
(33) An antibody that comprises a light chain variable region comprising the amino
acid substitutions of (28), (30), and (31).
(34) An antibody that comprises the heavy chain variable region of (32) and the light
chain variable region of (33).
(35) An antibody that comprises a heavy chain variable region comprising the amino
acid sequence of SEQ ID NO: 104 (VH of H53/L28).
(36) An antibody that comprises a light chain variable region comprising the amino
acid sequence of SEQ ID NO: 105 (VL of H53/L28).
(37) An antibody that comprises the heavy chain variable region of (35) and the light
chain variable region of (36).
[0177] Any amino acid substitutions of (1) to (37) described above are preferably introduced
into a humanized PM-1 antibody. The present invention provides antibodies comprising
at least the amino acid substitution of any one of (1) to (37) described above and
methods for producing those antibodies. Thus, the antibodies of the present invention
also include antibodies comprising other amino acid substitutions in addition to the
amino acid substitution of any one of (1) to (37) described above. The antibodies
of the present invention also include antibodies comprising combinations of multiple
amino acid substitutions of (1) to (37) described above. The amino acid substitutions
of (1) to (37) described above include, for example, substitutions in the amino acid
sequences of FR and CDR described above. Amino acid substitutions other than those
of (1) to (37) described above include other substitutions, deletions, additions,
and/or insertions in FR and CDR sequences than those described above. The amino acid
substitutions also include substitutions, deletions, additions, and/or insertions
in the amino acid sequences of constant regions.
[0178] Furthermore, in addition to those described above, amino acid alternations that result
in a lower isoelectric point without loss of the activity of anti-IL-6 receptor antibody,
include, for example, substitutions of Lys at position 15 and/or Ser at position 16
in the amino acid sequence of SEQ ID NO: 2 with other amino acids. The type of amino
acid after substitution is not particularly limited; however, substitutions of Gln
for Lys at position 15 and Asp for Ser at position 16 are preferred. A sequence comprising
the substitutions of Gln for Lys at position 15 and Asp for Ser at position 16 in
the amino acid sequence of SEQ ID NO: 2 is shown in SEQ ID NO: 121. Alternatively,
such amino acid substitutions may also be introduced into the amino acid sequence
of SEQ ID NO: 100. A sequence comprising the substitutions of Gln for Lys at position
15 and Asp for Gly at position 16 in the amino acid sequence of SEQ ID NO: 100 is
shown in SEQ ID NO: 122. Thus, the present invention provides antibodies that comprise
a heavy chain variable region comprising CDR2 in which Lys at position 15 and/or Ser
at position 16 in the amino acid sequence of SEQ ID NO: 2 or 100 have been substituted
with other amino acids.
[0179] Other alterations that result in a lower isoelectric point include substitution of
Gln at position 4 in the amino acid sequence of SEQ ID NO: 4 has been substituted
with another amino acid. The type of amino acid after substitution is not particularly
limited; however, substitution to Glu is preferred. An amino acid sequence comprising
the substitution of Glu for Gln at position 4 in the amino acid sequence of SEQ ID
NO: 4 is shown in SEQ ID NO: 123. Alternatively, this amino acid substitution may
also be introduced into the amino acid sequence of SEQ ID NO: 101. An amino acid sequence
comprising the substitution of Glu for Gln at position 4 in the amino acid sequence
of SEQ ID NO: 101 is shown in SEQ ID NO: 124. Thus, the present invention provides
antibodies that comprise a light chain variable region comprising CDR1 in which Gln
at position 4 in the amino acid sequence of SEQ ID NO: 4 or 101 has been substituted
with another amino acid.
[0180] Other alterations that result in a lower isoelectric point include substitution of
His at position 6 in the amino acid sequence of SEQ ID NO: 5 with another amino acid.
The type of amino acid after substitution is not particularly limited; however, substitution
to Glu is preferred. An amino acid sequence comprising the substitution of Glu for
His at position 6 in the amino acid sequence of SEQ ID NO: 5 is shown in SEQ ID NO:
125. Alternatively, this amino acid substitution may also be introduced into the amino
acid sequence of SEQ ID NO: 103. An amino acid sequence comprising the substitution
of Glu for His at position 6 in the amino acid sequence of SEQ ID NO: 103 is shown
in SEQ ID NO: 126. Thus, the present invention provides antibodies that comprise a
light chain variable region comprising CDR2 in which His at position 6 in the amino
acid sequence of SEQ ID NO: 5 or 103 has been substituted with another amino acid.
[0181] Furthermore, alternations that result in reduced immunogenicity risk include substitution
of Val for Ala at position 27 (H89 in Kabat's numbering system) in the amino acid
sequence of heavy chain FR3 of SEQ ID NO: 90. An amino acid sequence comprising the
substitution of Val for Ala at position 27 in the amino acid sequence of SEQ ID NO:
90 is shown in SEQ ID NO: 127. Thus, the present invention provides antibodies that
comprise a heavy chain variable region comprising FR3 in which Val has been substituted
for Ala at position 27 in the amino acid sequence of SEQ ID NO: 90.
[0182] The only mouse sequence that remains in the amino acid sequences of heavy chain FR3
of SEQ ID NO: 9 and 90 is Arg at position 6 (H71 in Kabat's numbering system). Anti-human
IL-6 receptor antibodies having a framework consisting entirely of human sequences
can be produced by using as a FR3 sequence, the human sequence of human VH1 subclass
(SEQ ID NO: 128) or human VH3 subclass (SEQ ID NO: 129) where Arg is conserved at
H71. Thus, the present invention provides antibodies that comprise a heavy chain variable
region comprising the FR3 of SEQ ID NO: 128 or 129.
[0183] Furthermore, alternations that improve stability include substitution of Ile for
Ser at position 5 (H107 in Kabat's numbering system) in the amino acid sequence of
heavy chain FR4 of SEQ ID NO: 10. An amino acid sequence comprising the substitution
of Ile for Ser at position 5 in the amino acid sequence of SEQ ID NO: 10 is shown
in SEQ ID NO: 130. Alternatively, this amino acid sequence may also be introduced
into the amino acid sequence of SEQ ID NO: 91. An amino acid sequence comprising the
substitution of Ile for Ser at position 5 in the amino acid sequence of SEQ ID NO:
91 is shown in SEQ ID NO: 131. Thus, the present invention provides antibodies that
comprise a heavy chain variable region comprising FR4 in which Ile has been substituted
for Ser at position 5 in the amino acid sequence of SEQ ID NO: 10 or 91.
[0184] Such amino acid substitutions are preferably introduced into the humanized PM-1 antibody,
H53/L28 (an antibody comprising the heavy chain variable region of SEQ ID NO: 104
and the light chain variable region of SEQ ID NO: 105), or PF1 antibody (an antibody
comprising the heavy chain variable region of SEQ ID NO: 22 and the light chain variable
region of SEQ ID NO: 23). The present invention provides antibodies comprising at
least such amino acid substitutions and methods for producing the antibodies. Thus,
the antibodies of the present invention include antibodies comprising, in addition
to such amino acid substitutions, the amino acid substitution of any one of (1) to
(37) described above and/or other amino acid substitutions than those of (1) to (37)
described above. Amino acid substitutions other than those of (1) to (37) described
above include other substitutions, deletions, additions, and/or insertions in FR and
CDR sequences than those described above. The amino acid substitutions also include
substitutions, deletions, additions, and/or insertions in the amino acid sequences
of constant regions.
<Anti-human IL-6 receptor antibodies with low isoelectric point>
[0185] The present invention also provides anti-IL-6 receptor antibodies with a low isoelectric
point. The antibodies of the present invention with low isoelectric point include
antibodies in which the measured isoelectric point of the whole antibody is low and
antibodies in which the theoretical isoelectric point of the variable region (VH/VL)
is low.
[0186] Herein, "anti-IL-6 receptor antibodies in which the measured isoelectric point of
the whole antibody is low" typically refers to antibodies in which the measured isoelectric
point is 7.5 or less, preferably 7.0 or less, and more preferably 6.0 or less. The
measured isoelectric point can be determined by methods known to those skilled in
the art, for example, non-denaturation gel isoelectric focusing or capillary isoelectric
focusing.
[0187] Herein, "anti-IL-6 receptor antibodies in which the theoretical isoelectric point
of the variable region is low" typically refers to antibodies in which the theoretical
isoelectric point is 5.5 or less, preferably 5.0 or less, and more preferably 4.0
or less. The theoretical isoelectric point can be determined by methods known to those
skilled in the art. For example, the theoretical isoelectric points of VH and VL of
a variable region can be computed by using software such as GENETYX (GENETYX CORPORATION).
[0188] There is no limitation on the type of amino acid substitution to be introduced to
obtain anti-IL-6 receptor antibodies of the present invention with low isoelectric
point. Such amino acid substitutions include, for example, the amino acid substitutions
described above. Such anti-IL-6 receptor antibodies with low isoelectric point are
assumed to show enhanced pharmacokinetics.
[0189] The type of IL-6 receptor is not particularly limited; however, human IL-6 receptor
is preferred.
<Anti-human IL-6 receptor antibodies that are stable at high concentrations>
[0190] Furthermore, the present invention provides anti-IL-6 receptor antibodies that are
stable at high concentrations.
[0191] Herein, "stable at high concentrations" means that the increase in the proportion
of aggregates of anti-IL-6 receptor antibody ([peak area for aggregate in gel filtration
chromatogram]/[total peak area in gel filtration chromatogram] x 100) generated in
a high-concentration antibody solution (100 mg/ml) at 25°C in one month is 0.3% or
less, preferably 0.2% or less, and more preferably 0.1% or less when the antibody
is in a buffer of pH 6.5 to 7.0 properly selected for subcutaneous administration,
for example, 20 mM histidine-HCl, 150 mM Nacl. The concentration of anti-IL-6 receptor
antibody may be 100 mg/ml or higher, for example, 200 or 300 mg/ml.
[0192] There is no limitation on the anti-IL-6 receptor antibodies of the present invention
that are stable at high concentrations. The antibodies can be prepared, for example,
with the above-described amino acid substitutions or such.
[0193] The type of IL-6 receptor is not particularly limited; however, human IL-6 receptor
is preferred.
[0194] The present invention also provides humanized PM-1 antibodies comprising any one
of the amino acid substitutions of (1) to (37) described above and further comprising
any of the amino acid substitutions of (a) to (y) described above to improve their
binding activity and/or neutralizing activity. In an embodiment, such antibodies include
those comprising a heavy chain variable region comprising the amino acid sequence
of SEQ ID NO: 22 (PF1_H) and a light chain variable region comprising the amino acid
sequence of SEQ ID NO: 23 (PF1_L) (PF1), but are not limited thereto.
[0195] Furthermore, the present invention provides anti-IL-6 receptor antibodies of any
of the following:
- (A) a heavy chain variable region that comprises CDR1 comprising the amino acid sequence
of SEQ ID NO: 165 (CDR1 of VH5-M83), CDR2 comprising the amino acid sequence of SEQ
ID NO: 166 (CDR2 of VH5-M83), and CDR3 comprising the amino acid sequence of SEQ ID
NO: 167 (CDR3 of VH5-M83);
- (B) a light chain variable region that comprises CDR1 comprising the amino acid sequence
of SEQ ID NO: 101 (CDR1 of VL5), CDR2 comprising the amino acid sequence of SEQ ID
NO: 168 (CDR2 of VL5), and CDR3 comprising the amino acid sequence of SEQ ID NO: 79
(CDR3 of VL5);
- (C) an antibody that comprises the heavy chain variable region of (A) and the light
chain variable region of (B);
- (D) a heavy chain variable region that comprises CDR1 comprising the amino acid sequence
of SEQ ID NO: 169 (CDR1 of VH3-M73), CDR2 comprising the amino acid sequence of SEQ
ID NO: 170 (CDR2 of VH3-M73), and CDR3 comprising the amino acid sequence of SEQ ID
NO: 171 (CDR3 of VH3-M73);
- (E) a light chain variable region that comprises CDR1 comprising the amino acid sequence
of SEQ ID NO: 172 (CDR1 of VL3), CDR2 comprising the amino acid sequence of SEQ ID
NO: 173 (CDR2 of VL3), and CDR3 comprising the amino acid sequence of SEQ ID NO: 79
(CDR3 of VL3);
- (F) an antibody that comprises the heavy chain variable region of (D) and the light
chain variable region of (E);
- (G) a heavy chain variable region that comprises CDR1 comprising the amino acid sequence
of SEQ ID NO: 169 (CDR1 of VH4-M73), CDR2 comprising the amino acid sequence of SEQ
ID NO: 174 (CDR2 of VH4-M73), and CDR3 comprising the amino acid sequence of SEQ ID
NO: 171 (CDR3 of VH4-M73);
- (H) a light chain variable region that comprises CDR1 comprising the amino acid sequence
of SEQ ID NO: 175 (CDR1 of VL1), CDR2 comprising the amino acid sequence of SEQ ID
NO: 173 (CDR2 of VL1), and CDR3 comprising the amino acid sequence of SEQ ID NO: 79
(CDR3 of VL1); and
- (I) an antibody that comprises the heavy chain variable region of (G) and the light
chain variable region of (H).
[0196] Furthermore, the present invention provides anti-IL-6 receptor antibodies of any
of the following:
- (a) an antibody that comprises a heavy chain variable region comprising the amino
acid sequence of SEQ ID NO: 159 (H96-IgG1 variable region);
- (b) an antibody that comprises a heavy chain variable region in which at least one
of amino acids of Trp at position 35, Tyr at position 51, Ser at position 63, Lys
at position 65, Gly at position 66, Val at position 99, Ile at position 103, Tyr at
position 108, Glu at position 111, and Thr at position 113 in the amino acid sequence
of SEQ ID NO: 159 (H96-IgG1 variable region) has been substituted with another amino
acid;
- (c) an antibody that comprises a heavy chain variable region comprising an amino acid
sequence in which Lys at position 65, Gly at position 66, Val at position 99, IIe
at position 103, Glu at position 111, and Thr at position 113 in the amino acid sequence
of SEQ ID NO: 159 (H96-IgG1 variable region) have been substituted with other amino
acids;
- (d) an antibody that comprises a heavy chain variable region comprising an amino acid
sequence in which Trp at position 35, Tyr at position 51, Ser at position 63, Lys
at position 65, Gly at position 66, Val at position 99, Ile at position 103, and Tyr
at position 108 in the amino acid sequence of SEQ ID NO: 159 (H96-IgG1 variable region)
have been substituted with other amino acids;
- (e) an antibody that comprises a heavy chain variable region comprising the amino
acid sequence of SEQ ID NO: 160 (F2H-IgG1 variable region);
- (f) an antibody that comprises a heavy chain variable region comprising the amino
acid sequence of SEQ ID NO: 161 (VH5-M83 variable region);
- (g) an antibody that comprises a light chain variable region comprising an amino acid
sequence in which Gln at position 27 and/or His at position 55 in the amino acid sequence
of SEQ ID NO: 23 (PF1L) have been substituted with other amino acids;
- (h) an antibody that comprises a light chain variable region comprising the amino
acid sequence of SEQ ID NO: 162 (L39 variable region);
- (i) an antibody that comprises a light chain variable region comprising the amino
acid sequence of SEQ ID NO: 163 (VL5-kappa variable region);
- (j) an antibody that comprises a heavy chain variable region comprising the amino
acid sequence of SEQ ID NO: 176 (VH3-M73 variable region);
- (k) an antibody that comprises a heavy chain variable region comprising the amino
acid sequence of SEQ ID NO: 178 (VH4-M73 variable region);
- (l) an antibody that comprises a light chain variable region comprising the amino
acid sequence of SEQ ID NO: 177 (VL3-kappa variable region);
- (m) an antibody that comprises a light chain variable region comprising the amino
acid sequence of SEQ ID NO: 179 (VL1-kappa variable region);
- (n) an antibody that comprises the heavy chain variable region of (e) and the light
chain variable region of (h);
- (o) an antibody that comprises the heavy chain variable region of (f) and the light
chain variable region of (i) (combination of FV5-M83 variable regions);
- (p) an antibody that comprises the heavy chain variable region of (j) and the light
chain variable region of (l) (combination of FV4-M73 variable regions); and
- (q) an antibody that comprises the heavy chain variable region of (k) and the light
chain variable region of (m) (combination of FV3-M73 variable regions).
[0197] The type of amino acid after substitution is not particularly limited in the amino
acid substitution of the heavy chain variable regions of (a) to (d) above; however,
substitutions of Val for Trp at position 35, Phe for Tyr at position 51, Thr for Ser
at position 63, Gln for Lys at position 65, Asp for Gly at position 66, Leu for Val
at position 99, Ala for Ile at position 103, Val for Tyr at position 108, Gln for
Glu at position 111, Ile for Thr at position 113 are preferred. Alternatively, the
type of amino acid after substitution is not particularly limited in the amino acid
substitution of the light chain variable region of (g) above; however, substitutions
of Glu for Gln at position 27 and Glu for His at position 55 are preferred. Amino
acid substitutions, deletions, insertions, and/or additions other than the amino acid
substitution described above may be included.
[0198] The antibody constant regions of the present invention are not particularly limited,
and any constant regions may be used. For example, constant regions comprising a natural
sequence such as IgG1, IgG2, and IgG4 and altered constant regions prepared by introducing
amino acid substitutions, deletions, additions, and/or insertions into a constant
region comprising a natural sequence can be used. The examples of such altered constant
regions include the constant regions described below.
[0199] The examples of antibodies using the variable regions of the present invention mentioned
above include:
- (1) an antibody that comprises a heavy chain comprising the amino acid sequence of
SEQ ID NO: 134 (H96-IgG1);
- (2) an antibody that comprises a heavy chain comprising the amino acid sequence of
SEQ ID NO: 135 (F2H-IgG1);
- (3) an antibody that comprises a heavy chain comprising the amino acid sequence of
SEQ ID NO: 137 (VH5-IgG1);
- (4) an antibody that comprises a heavy chain comprising the amino acid sequence of
SEQ ID NO: 139 (VH5-M83);
- (5) an antibody that comprises a heavy chain comprising the amino acid sequence of
SEQ ID NO: 136 (L39);
- (6) an antibody that comprises a heavy chain comprising the amino acid sequence of
SEQ ID NO: 138 (VL5-kappa);
- (7) an antibody that comprises a heavy chain comprising the amino acid sequence of
SEQ ID NO: 180 (VH3-M73);
- (8) an antibody that comprises a heavy chain comprising the amino acid sequence of
SEQ ID NO: 182 (VH4-M73);
- (9) an antibody that comprises a light chain comprising the amino acid sequence of
SEQ ID NO: 181 (VL3-kappa);
- (10) an antibody that comprises a light chain comprising the amino acid sequence of
SEQ ID NO: 183 (VL1-kappa);
- (11) an antibody that comprises the heavy chain of (2) and the light chain of (5);
- (12) an antibody that comprises the heavy chain of (3) and the light chain of (6);
- (13) an antibody that comprises the heavy chain of (4) and the light chain of (6)
(FV5-M83);
- (14) an antibody that comprises the heavy chain of (7) and the light chain of (9)
(FV4-M73);
- (15) an antibody that comprises the heavy chain of (8) and the light chain of (10)
(FV3-M73); and
- (16) an antibody having an activity equivalent to that of any of the antibodies of
(1) to (15).
[0200] Herein, "having equivalent activity" means that the antigen binding activity and/or
neutralizing activity are equivalent. "Equivalent activity" in the present invention
does not necessarily mean completely identical activity, but may be, for example,
50% or more of the activity, preferably 70% or more, and more preferably 90% or more.
[0201] Furthermore, the present invention provides CDR and FR of any of the following:
- (i) a heavy chain FR1 that comprises the amino acid sequence of SEQ ID NO: 84 (heavy
chain FR1 of VH5);
- (ii) a heavy chain FR1 that comprises the amino acid sequence of SEQ ID NO: 186 (heavy
chain FR1 of VH3 and VH4);
- (iii) a heavy chain FR2 that comprises the amino acid sequence of SEQ ID NO: 85 (heavy
chain FR2 of VH3, VH4, and VH5);
- (iv) a heavy chain FR3 that comprises the amino acid sequence of SEQ ID NO: 184 (heavy
chain FR3 of VH3, VH4, and VH5);
- (v) a heavy chain FR4 that comprises the amino acid sequence of SEQ ID NO: 133 (heavy
chain FR4 of VH3, VH4, and VH5);
- (vi) a light chain FR1 that comprises the amino acid sequence of SEQ ID NO: 92 (light
chain FR1 of VL1, VL3, and VL5);
- (vii) a light chain FR2 that comprises the amino acid sequence of SEQ ID NO: 93 (light
chain FR2 af VL1, VL3, and VL5);
- (viii) a light chain FR3 that comprises the amino acid sequence of SEQ ID NO: 97 (light
chain FR3 of VL1, VL3, and VL5);
- (ix) a light chain FR4 that comprises the amino acid sequence of SEQ ID NO: 98 (light
chain FR4 of VL1, VL3, and VL5);
- (x) a heavy chain CDR1 that comprises the amino acid sequence of SEQ ID NO: 169 (heavy
chain CDR1 of VH3 and VH4);
- (xi) a heavy chain CDR1 that comprises the amino acid sequence of SEQ ID NO: 165 (heavy
chain CDR1 of VH5);
- (xii) a heavy chain CDR2 that comprises the amino acid sequence of SEQ ID NO: 170
(heavy chain CDR2 of VH3);
- (xiii) a heavy chain CDR2 that comprises the amino acid sequence of SEQ ID NO: 174
(heavy chain CDR2 of VH4);
- (xiv) a heavy chain CDR2 that comprises the amino acid sequence of SEQ ID NO: 166
(heavy chain CDR2 of VH5);
- (xv) a heavy chain CDR3 that comprises the amino acid sequence of SEQ ID NO: 171 (heavy
chain CDR3 of VH3 and VH4);
- (xvi) a heavy chain CDR3 that comprises the amino acid sequence of SEQ ID NO: 167
(heavy chain CDR3 of VH5);
- (xvii) a light chain CDR1 that comprises the amino acid sequence of SEQ ID NO: 175
(light chain CDR1 of VL1;
- (xviii) a light chain CDR1 that comprises the amino acid sequence of SEQ ID NO: 172
(light chain CDR1 of VL3);
- (xix) a light chain CDR1 that comprises the amino acid sequence of SEQ ID NO: 101
(light chain CDR1 of VL5);
- (xx) a light chain CDR2 that comprises the amino acid sequence of SEQ ID NO: 173 (light
chain CDR2 of VL1 and VL3);
- (xxi) a light chain CDR2 that comprises the amino acid sequence of SEQ ID NO: 168
(light chain CDR2 of VL5); and
- (xxii) a light chain CDR3 that comprises the amino acid sequence of SEQ ID NO: 79
(light chain CDR3 of VL1, VL3, and VL5).
[0202] The antibodies of the present invention also include fragments and processed products
of antibodies comprising any of the amino acid substitutions described above. Such
antibody fragments include, for example, Fab, F(ab')2, Fv, single chain Fv (scFv)
in which H and L chains are linked together via an appropriate linker, single domain
H chain and single domain L chain (for example,
Nat. Biotechnol. 2005 Sep;23(9):1126-36), Unibody (
WO 2007059782 A1), and SMIP (
WO 2007014278 A2). The origin of antibodies is not particularly limited. The antibodies include human,
mouse, rat, and rabbit antibodies. The antibodies of the present invention may be
chimeric, humanized, fully humanized antibodies, or such.
[0203] Specifically, such antibody fragments are obtained by treating antibodies with an
enzyme, for example, papain or pepsin, or by constructing genes to encode such antibody
fragments, inserting them into expression vectors, and then expressing them in appropriate
host cells (see, for example,
Co, M. S. et al., J. Immunol. (1994) 152, 2968-2976;
Better, M. and Horwitz, A. H. Methods in Enzymology (1989) 178, 476-496;
Plückthun, A.; Skerra, A., Methods in Enzymology (1989) 178, 497-515;
Lamoyi, E., Methods in Enzymology (1989) 121, 652-663;
Rousseaux, J. et al., Methods in Enzymology (1989) 121, 663-66;
Bird, R. E. et al., TIBTECH (1991) 9, 132-137).
[0204] scFv is obtained by linking V regions of antibody H and L chains. In such scFv, the
H chain V region is linked to the L chain V region via a linker, preferably a peptide
linker (
Huston, J. S. et al., Proc. Natl. Acad. Sci. USA (1988) 85, 5879-5883). The H chain and L chain V regions in an scFv may be derived from any of the antibodies
described above. The peptide linker to link the V regions includes, for example, arbitrary
single chain peptides of 12 to 19 amino acid residues.
<Antibody constant regions>
[0205] The present invention also provides the antibody constant regions of (i) to (xxi)
described below, which have been improved through amino acid substitution. The constant
region refers to IgG1, IgG2, or IgG4 type constant region. The amino acid sequences
of human IgG1, IgG2, and IgG4 constant regions are known (human IgG1 constant region,
SEQ ID NO: 19; human IgG2 constant region, SEQ ID NO: 20; and human IgG4 constant
region, SEQ ID NO: 21). The sequence of human IgG4 constant region has been altered
to improve the stability of the hinge region (
Mol. Immunol, 1993 Jan;30(1):105-8). The present invention also provides antibodies that comprise such an amino acid
substitution-containing antibody constant region. The antibody constant regions are
preferably human antibody constant regions.
[0206] The amino acid substitution-containing antibody constant regions of the present invention
may comprise other amino acid substitutions or modifications as long as they comprise
the amino acid substitution of any one of (i) to (xxi) described below. Therefore,
IgG2 constant regions comprising the amino acid substitutions of the present invention
in the IgG2 constant region comprising the amino acid sequence of SEQ ID NO: 20 include
IgG2 constant regions that comprise one or more amino acid substitutions and/or modifications
in the amino acid sequence of SEQ ID NO: 20 and further comprise the amino acid substitutions
of the present invention, as well as IgG2 constant regions that comprise the amino
acid substitutions of the present invention and further comprise one or more amino
acid substitutions and/or modifications. The same applies to IgG1 constant regions
comprising the amino acid sequence of SEQ ID NO: 19 and IgG4 constant regions comprising
the amino acid sequence of SEQ ID NO: 21.
[0207] Furthermore, the sugar chain at position 297 in the EU numbering system (see sequences
of proteins of immunological interest, NIH Publication No.91-3242) may be of any sugar-chain
structure, or there may not be any sugar chain linked at this site (for example, constant
regions produced in host cells where glycosylation does not occur, such as
E.
coli).
(i) Improvement of the stability of IgG2 constant region at acidic conditions
[0208] In an embodiment, the IgG2 constant region of the present invention comprising amino
acid substitutions includes IgG2 constant regions in which Met at position 276 (position
397 in the EU numbering system) in the amino acid sequence of SEQ ID NO: 20 has been
substituted with another amino acid. The type of amino acid after substitution is
not particularly limited; however, substitution to Val is preferred. The antibody
stability under acidic conditions can be improved by substituting Met at position
276 (position 397 in the EU numbering system) in the amino acid sequence of SEQ ID
NO: 20 with another amino acid.
(ii) Improvement of the heterogeneity of IgG2 constant region
[0209] In an embodiment, the IgG2 constant region of the present invention comprising amino
acid substitutions includes IgG2 constant regions in which Cys at position 14 (position
131 in the EU numbering system), Arg at position 16 (position 133 in the EU numbering
system), and Cys at position 102 (position 219 in the EU numbering system) in the
amino acid sequence of SEQ ID NO: 20 have been substituted with other amino acids.
The type of amino acid after substitution is not particularly limited; however, substitutions
of Ser for Cys at position 14 (position 131 in the EU numbering system), Lys for Arg
at position 16 (position 133 in the EU numbering system), and Ser for Cys at position
102 (position 219 in the EU numbering system) (IgG2-SKSC) are preferred.
[0210] These substitutions can reduce the heterogeneity originated from the hinge region
of IgG2. The IgG2 constant regions of the present invention comprising amino acid
substitutions include IgG2 constant regions comprising at least one of the three types
of amino acid substitutions described above; however, the IgG2 constant regions preferably
comprise substitutions of Cys at position 14 and Cys at position 102 with other amino
acids or all three types of the amino acid substitutions described above.
(iii) Impairment of the binding of IgG2 constant region to FcγR
[0211] In an embodiment, the present invention also provides IgG2 constant regions comprising
an amino acid sequence in which Ala at position 209 (EU330), Pro at position 210 (EU331),
and/or Thr at position 218 (EU339) of the amino acid sequence of SEQ ID NO: 20 have
been substituted with Ser, Ser, and Ala, respectively. The substitutions for Ala at
position 209 (EU330) and for Pro at position 210 (EU331) have already been reported
to enable the impairment of the Fcγ receptor binding (
Eur. J. Immunol. 1999 Aug;29(8):2613-24). From the perspective of immunogenicity risk, however, these alterations are not
preferred because they result in generation of non-human derived peptides that can
become T-cell epitopes. However, the Fcγ receptor binding of IgG2 can be reduced by
substituting Ala for Thr at position 218 (EU339) at the same time, and the 9-12 amino
acid peptides which can become T-cell epitopes are derived from human only.
[0212] The IgG2 constant regions of the present invention comprising amino acid substitutions
comprise at least one of the three types of amino acid substitutions described above;
however, the IgG2 constant regions preferably comprise all three types of the amino
acid substitutions described above. In a preferred embodiment, the IgG2 constant regions
of the present invention comprising amino acid substitutions include IgG2 constant
regions comprising an amino acid sequence in which Ala at position 209 (EU330), Pro
at position 210 (EU331), and Thr at position 218 (EU339) in the amino acid sequence
of SEQ ID NO: 20 have been substituted with Ser, Ser, and Ala, respectively.
(iv) Improvement of the C-terminal heterogeneity of IgG2 constant region
[0213] The present invention provides IgG2 constant regions comprising an amino acid sequence
in which Gly at position 325 (position 446 in the EU numbering system) and Lys at
position 326 (position 447 in the EU numbering system) have been deleted in the amino
acid sequence of SEQ ID NO: 20. The heterogeneity originated from the C terminus of
antibody H chain can be reduced only when both of the amino acids are deleted.
(v) Improvement of the pharmacokinetics by altering IgG2 constant region
[0214] An embodiment of the IgG2 constant regions with amino acid substitutions of the present
invention includes IgG2 constant regions in which His at position 147 (position 268
in the EU numbering system), Arg at position 234 (position 355 in the EU numbering
system), and Gln at position 298 (position 419 in the EU numbering system) in the
amino acid sequence of SEQ ID NO: 20 have been substituted with other amino acids.
These amino acid substitutions enable to improve antibody pharmacokinetics. The type
of amino acid after substitution is not particularly limited; however, substitutions
of Gln for His at position 147 (position 268 in the EU numbering system), Gln for
Arg at position 234 (position 355 in the EU numbering system), and Glu for Gln at
position 298 (position 419 in the EU numbering system) are preferred. The IgG2 constant
regions with amino acid substitutions of the present invention include IgG2 constant
regions comprising at least one of the three types of the amino acid substitutions
described above; however, the IgG2 constant regions preferably comprise all three
types of the amino acid substitutions described above.
(vi) Improvement of the stability of IgG4 constant region at acidic conditions
[0215] The present invention provides IgG4 constant regions comprising an amino acid sequence
in which Arg at position 289 (position 409 in the EU numbering system) of the amino
acid sequence of SEQ ID NO: 21 has been substituted with another amino acid. The type
of amino acid after substitution is not particularly limited; however, substitution
to Lys is preferred. The antibody stability under acidic conditions can be improved
by substituting Arg at position 289 (position 409 in the EU numbering system) in the
amino acid sequence of SEQ ID NO: 21 with another amino acid.
(vii) Improvement of the C-terminal heterogeneity of IgG4 constant region
[0216] The present invention provides IgG4 constant regions comprising an amino acid sequence
in which Gly at position 326 (position 446 in the EU numbering system) and Lys at
position 327 (position 447 in the EU numbering system) have been deleted in the amino
acid sequence of SEQ ID NO: 21. The heterogeneity originated from the C terminus of
antibody H chain can be reduced only when both of the amino acids are deleted.
(viii) Improvement of the C-terminal heterogeneity of IgG1 constant region
[0217] The present invention provides IgG1 constant regions comprising an amino acid sequence
in which Gly at position 329 (position 446 in the EU numbering system) and Lys at
position 330 (position 447 in the EU numbering system) have been deleted in the amino
acid sequence of SEQ ID NO: 19. The heterogeneity originated from the C terminus of
antibody H chain can be reduced only when both of the amino acids are deleted.
(ix)
[0218] The present invention provides IgG1 constant regions comprising an amino acid sequence
in which Asn at position 317 (position 434 in the EU numbering system) in the amino
acid sequence of SEQ ID NO: 19 has been substituted with another amino acid.
[0219] The type of amino acid after substitution is not particularly limited; however, substitution
to Ala is preferred.
(x)
[0220] The present invention provides IgG2 constant regions comprising an amino acid sequence
in which Ala at position 209 (position 330 in the EU numbering system), Pro at position
210 (position 331 in the EU numbering system), Thr at position 218 (position 339 in
the EU numbering system), Met at position 276 (position 397 in the EU numbering system),
Cys at position 14 (position 131 in the EU numbering system), Arg at position 16 (position
133 in the EU numbering system), Cys at position 102 (position 219 in the EU numbering
system), Glu at position 20 (position 137 in the EU numbering system), and Ser at
position 21 (position 138 in the EU numbering system) in the amino acid sequence of
SEQ ID NO: 20 have been substituted with other amino acids.
[0221] The type of amino acid after substitution is not particularly limited; however, substitutions
of Ser for Ala at position 209, Ser for Pro at position 210, Ala for Thr at position
218, Val for Met at position 276, Ser for Cys at position 14, Lys for Arg at position
16, Ser for Cys at position 102, Gly for Glu at position 20, and Gly for Ser at position
21 are preferred.
(xi)
[0222] The present invention provides IgG2 constant regions comprising an amino acid sequence
in which Ala at position 209 (position 330 in the EU numbering system), Pro at position
210 (position 331 in the EU numbering system), Thr at position 218 (position 339 in
the EU numbering system), Met at position 276 (position 397 in the EU numbering system),
Cys at position 14 (position 131 in the EU numbering system), Arg at position 16 (position
133 in the EU numbering system), Cys at position 102 (position 219 in the EU numbering
system), Glu at position 20 (position 137 in the EU numbering system), and Ser at
position 21 (position 138 in the EU numbering system) have been substituted with other
amino acids, and simultaneously Gly at position 325 (position 446 in the EU numbering
system) and Lys at position 326 (position 447 in the EU numbering system) have been
deleted in the amino acid sequence of SEQ ID NO: 20.
[0223] The type of amino acid after substitution is not particularly limited; however, substitutions
of Ser for Ala at position 209, Ser for Pro at position 210, Ala for Thr at position
218, Val for Met at position 276, Ser for Cys at position 14, Lys for Arg at position
16, Ser for Cys at position 102, Gly for Glu at position 20, and Gly for Ser at position
21 are preferred.
(xii)
[0224] The present invention provides IgG2 constant regions comprising an amino acid sequence
in which Met at position 276 (position 397 in the EU numbering system), Cys at position
14 (position 131 in the EU numbering system), Arg at position 16 (position 133 in
the EU numbering system), Cys at position 102 (position 219 in the EU numbering system),
Glu at position 20 (position 137 in the EU numbering system), and Ser at position
21 (position 138 in the EU numbering system) in the amino acid sequence of SEQ ID
NO: 20 have been substituted with other amino acids.
[0225] The type of amino acid after substitution is not particularly limited; however, substitutions
of Val for Met at position 276, Ser for Cys at position 14, Lys for Arg at position
16, Ser for Cys at position 102, Gly for Glu at position 20, and Gly for Ser at position
21 are preferred.
(xiii)
[0226] The present invention provides IgG2 constant regions comprising an amino acid sequence
in which Met at position 276 (position 397 in the EU numbering system), Cys at position
14 (position 131 in the EU numbering system), Arg at position 16 (position 133 in
the EU numbering system), Cys at position 102 (position 219 in the EU numbering system),
Glu at position 20 (position 137 in the EU numbering system), and Ser at position
21 (position 138 in the EU numbering system) have been substituted with other amino
acids, and simultaneously Gly at position 325 (position 446 in the EU numbering system)
and Lys at position 326 (position 447 in the EU numbering system) have been deleted
in the amino acid sequence of SEQ ID NO: 20.
[0227] The type of amino acid after substitution is not particularly limited; however, substitutions
of Val for Met at position 276, Ser for Cys at position 14, Lys for Arg at position
16, Ser for Cys at position 102, Gly for Glu at position 20, and Gly for Ser at position
21 are preferred.
(xiv)
[0228] The present invention provides IgG2 constant regions comprising an amino acid sequence
in which Cys at position 14 (position 131 in the EU numbering system), Arg at position
16 (position 133 in the EU numbering system), Cys at position 102 (position 219 in
the EU numbering system), Glu at position 20 (position 137 in the EU numbering system),
Ser at position 21 (position 138 in the EU numbering system), His at position 147
(position 268 in the EU numbering system), Arg at position 234 (position 355 in the
EU numbering system), and Gin at position 298 (position 419 in the EU numbering system)
have been substituted with other amino acids, and simultaneously Gly at position 325
(position 446 in the EU numbering system) and Lys at position 326 (position 447 in
the EU numbering system) have been deleted in the amino acid sequence of SEQ ID NO:
20.
[0229] The type of amino acid after substitution is not particularly limited; however, substitutions
of Ser for Cys at position 14, Lys for Arg at position 16, Ser for Cys at position
102, Gly for Glu at position 20, Gly for Ser at position 21, Gln for His at position
147, Gln for Arg at position 234, and Glu for Gln at position 298 are preferred.
(xv)
[0230] The present invention provides IgG2 constant regions comprising an amino acid sequence
in which Cys at position 14 (position 131 in the EU numbering system), Arg at position
16 (position 133 in the EU numbering system), Cys at position 102 (position 219 in
the EU numbering system), Glu at position 20 (position 137 in the EU numbering system),
Ser at position 21 (position 138 in the EU numbering system), His at position 147
(position 268 in the EU numbering system), Arg at position 234 (position 355 in the
EU numbering system), Gln at position 298 (position 419 in the EU numbering system),
and Asn at position 313 (position 434 in the EU numbering system) have been substituted
with other amino acids, and simultaneously Gly at position 325 (position 446 in the
EU numbering system) and Lys at position 326 (position 447 in the EU numbering system)
have been deleted in the amino acid sequence of SEQ ID NO: 20.
[0231] The type of amino acid after substitution is not particularly limited; however, substitutions
of Ser for Cys at position 14, Lys for Arg at position 16, Ser for Cys at position
102, Gly for Glu at position 20, Gly for Ser at position 21, Gln for His at position
147, Gln for Arg at position 234, Glu for Gln at position 298, and Ala for Asn at
position 313 are preferred.
(xvi)
[0232] The present invention provides IgG4 constant regions comprising an amino acid sequence
in which Arg at position 289 (position 409 in the EU numbering system), Cys at position
14, Arg at position 16, Glu at position 20, Ser at position 21, Arg at position 97,
Ser at position 100, Tyr at position 102, Gly at position 103, Pro at position 104,
and Pro at position 105 (positions 131, 133, 137, 138, 214, 217, 219, 220, 221, and
222 in the EU numbering system, respectively), Glu at position 113, Phe at position
114, and Leu at position 115 (positions 233, 234, and 235 in the EU numbering system,
respectively) have been substituted with other amino acids, and simultaneously Gly
at position 116 (position 236 in the EU numbering system) has been deleted in the
amino acid sequence of SEQ ID NO: 21.
[0233] The type of amino acid after substitution is not particularly limited; however, substitutions
of Ser for Cys at position 14 (position 131 in the EU numbering system), Lys for Arg
at position 16 (position 133 in the EU numbering system), Gly for Glu at position
20 (position 137 in the EU numbering system), Gly for Ser at position 21 (position
138 in the EtJ numbering system), Thr for Arg at position 97 (position 214 in the
EU numbering system), Arg for Ser at position 100 (position 217 in the EU numbering
system), Ser for Tyr at position 102 (position 219 in the EU numbering system), Cys
for Gly at position 103 (position 220 in the EU numbering system), Val for Pro at
position 104 (position 221 in the EU numbering system), Glu for Pro at position 145
(position 222 in the EU numbering system), Pro for Glu at position 113 (position 233
in the EU numbering system), Val for Phe at position 114 (position 234 in the EU numbering
system), Ala for Leu at position 115 (position 235 in the EU numbering system), and
Lys for Arg at position 289 (position 409 in the EU numbering system) are preferred.
(xvii)
[0234] The present invention provides IgG4 constant regions comprising an amino acid sequence
in which Arg at position 289 (position 409 in the EU numbering system), Cys at position
14, Arg at position 16, Glu at position 20, Ser at position 21, Arg at position 97,
Ser at position 144, Tyr at position 102, Gly at position 143, Pro at position 104,
and Pro at position 105 (positions 131, 133, 137, 138, 214, 217, 219, 220, 221, and
222 in the EU numbering system, respectively), Glu at position 113, Phe at position
114, and Leu at position 115 (positions 233, 234, and 235 in the EU numbering system,
respectively) have been substituted with other amino acids, and simultaneously Gly
at position 116 (position 236 in the EU numbering system), Gly at position 326 (position
446 in the EU numbering system), and Lys at position 327 (position 447 in the EU numbering
system) have been deleted in the amino acid sequence of SEQ ID NO: 21.
[0235] The type of amino acid after substitution is not particularly limited; however, substitutions
of Ser for Cys at position 14 (position 131 in the EU numbering system), Lys for Arg
at position 16 (position 133 in the EU numbering system), Gly for Glu at position
20 (position 137 in the EU numbering system), Gly for Ser at position 21 (position
138 in the EU numbering system), Thr for Arg at position 97 (position 214 in the EU
numbering system), Arg for Ser at position 100 (position 217 in the EU numbering system),
Ser for Tyr at position 102 (position 219 in the EU numbering system), Cys for Gly
at position 103 (position 220 in the EU numbering system), Val for Pro at position
104 (position 221 in the EU numbering system), Glu for Pro at position 105 (position
222 in the EU numbering system), Pro for Glu at position 113 (position 233 in the
EU numbering system), Val for Phe at position 114 (position 234 in the EU numbering
system), Ala for Leu at position 115 (position 235 in the EU numbering system), and
Lys for Arg at position 289 (position 409 in the EU numbering system) are preferred.
(xviii)
[0236] The present invention provides IgG1 constant regions comprising an amino acid sequence
in which Asn at position 317 (position 434 in the EU numbering system) has been substituted
with another amino acid, and simultaneously Gly at position 329 (position 446 in the
EU numbering system) and Lys at position 330 (position 447 in the EU numbering system)
have been deleted in the amino acid sequence of SEQ ID NO: 19.
[0237] The type of amino acid after substitution of Asn at position 317 (position 434 in
the EU numbering system) is not particularly limited; however, substitution to Ala
is preferred.
(xix)
[0238] Below is a preferred embodiment of IgG2 of the present invention, which has reduced
heterogeneity in the hinge region and/or reduced Fcγ receptor-binding activity.
[0239] Antibodies comprising an IgG2 constant region comprising an amino acid sequence in
which Ala at position 209, Pro at position 210, Thr at position 218, Cys at position
14, Arg at position 16, Cys at position 102, Glu at position 20, and Ser at position
21 in the amino acid sequence of SEQ ID NO: 20 have been substituted with other amino
acids.
[0240] The type of amino acid after substitution is not particularly limited; however, substitutions
of Ser for Ala at position 209 (position 330 in the EU numbering system), Ser for
Pro at position 210 (position 331 in the EU numbering system), Ala for Thr at position
218 (position 339 in the EU numbering system), Ser for Cys at position 14 (position
131 in the EU numbering system), Lys for Arg at position 16 (position 133 in the EU
numbering system), Ser for Cys at position 102 (position 219 in the EU numbering system),
Gly for Glu at position 20 (position 137 in the EU numbering system), and Gly for
Ser at position 21 (position 138 in the EU numbering system) are preferred.
[0241] Such IgG2 constant regions include, for example, IgG2 constant regions comprising
the amino acid sequence of SEQ ID NO: 191 (M86).
[0242] In another preferred embodiment, IgG2 constant regions of the present invention include
IgG2 constant regions resulting from the deletion of Gly at position 325 and Lys at
position 326 in the above-described IgG2 constant regions to reduce C-terminal heterogeneity.
Such antibodies include, for example, IgG2 that comprises a constant region comprising
the amino acid sequence of SEQ ID NO: 192 (M86ΔGK).
(xx)
[0243] Below is another preferred embodiment of the IgG2 constant regions of the present
invention, which have reduced heterogeneity in the hinge region.
[0244] IgG2 constant regions comprising an amino acid sequence in which Cys at position
14, Arg at position 16, Cys at position 102, Glu at position 20, and Ser at position
21 in the amino acid sequence of SEQ ID NO: 20 have been substituted with other amino
acids.
[0245] The type of amino acid after substitution is not particularly limited; however, substitutions
of Ser for Cys at position 14 (position 131 in the EU numbering system), Lys for Arg
at position 16 (position 133 in the EU numbering system), Ser for Cys at position
102 (position 219 in the EU numbering system), Gly for Glu at position 20 (position
137 in the EU numbering system), and Gly for Ser at position 21 (position 138 in the
EU numbering system) are preferred.
[0246] Such IgG2 constant regions include, for example, IgG2 constant regions comprising
the amino acid sequence of SEQ ID NO: 193 (M40).
[0247] In another preferred embodiment, the IgG2 constant regions of the present invention
include IgG2 constant regions further comprising the deletion of Gly at position 325
and Lys at position 326 in the above-described IgG2 constant regions. Such antibodies
include, for example, IgG2 constant regions comprising the amino acid sequence of
SEQ ID NO: 194 (M40ΔGK).
(xxi) M14ΔGK, M17ΔGK, M11ΔGK, M31ΔGK, M58, M73, M83, M86ΔGK, and M40ΔGK
[0248] The present invention also provides an antibody constant region comprising the amino
acid sequence of SEQ ID NO: 24 (M14ΔGK). The present invention also provides an antibody
constant region comprising the amino acid sequence of SEQ ID NO: 116 (M17ΔGK). The
present invention also provides an antibody constant region comprising the amino acid
sequence of SEQ ID NO: 25 (M11ΔGK). The present invention further provides an antibody
constant region comprising the amino acid sequence of SEQ ID NO: 118 (M31ΔGK). The
present invention further provides an antibody constant region comprising the amino
acid sequence of SEQ ID NO: 151 (M58). The present invention further provides an antibody
constant region comprising the amino acid sequence of SEQ ID NO: 153 (M73). The present
invention also provides an antibody constant region comprising the amino acid sequence
of SEQ ID NO: 164 (M83). The present invention further provides an antibody constant
region comprising the amino acid sequence of SEQ ID NO: 192 (M86ΔGK). The present
invention further provides an antibody constant region comprising the amino acid sequence
of SEQ ID NO: 194 (M40ΔGK). These antibody constant regions have been optimized to
have reduced Fcγ receptor binding activity, reduced immunogenicity risk, improved
stability under acidic conditions, reduced heterogeneity, improved pharmacokinetics,
and/or higher stability in preparations in comparison with the IgG1 constant region.
[0249] The present invention provides antibodies comprising the antibody constant region
of any one of (i) to (xxi) described above. There is no limitation on the type of
antigen and origin of antibody, as long as the antibodies comprise an antibody constant
region described above. The preferred antibodies include, for example, antibodies
that bind to IL-6 receptor. Alternatively, the preferred antibodies include, for example,
humanized antibodies. Such antibodies include, for example, antibodies comprising
the variable region of humanized PM-1 antibody. Such a variable region of humanized
PM-1 antibody may comprise any of the above-described amino acid substitutions, or
other amino acid substitutions, deletions, additions, and/or insertions. Specifically,
the substitutions include, for example, alterations that improve the affinity of (a)
to (y) described above; alterations that lower the isoelectric point of (i) to (viii)
described above, alternations that improve the stability of (α) to (ζ) described below;
and alternations that reduce immunogenicity, but are not limited thereto.
[0250] In one embodiment, such antibodies include antibodies that comprise a heavy chain
variable region comprising the amino acid sequence of SEQ ID NO: 113 (PF_1+M14ΔGK)
and a light chain variable region comprising the amino acid sequence of SEQ ID NO:
23 (PF1_L) (the light chain constant region may be kappa or lambda, or an altered
form thereof) (PF1), but are not limited thereto.
[0251] Alternatively, the antibody constant regions described above and/or antibody molecules
comprising an antibody constant region described above can be linked as a form of
Fc fusion molecule to antibody-like binding molecule (scaffold molecules), bioactive
peptides, binding peptides, or such.
[0252] The antibodies of the present invention can also be obtained by, for example, the
following methods in addition to those described in the Examples. In one embodiment
to obtain antibodies of the present invention, one or more amino acid residues are
first substituted with amino acids of interest in at least one region selected from
the group consisting of CDR, FR, and constant regions of an anti-IL-6 receptor antibody
known to those skilled in the art. Methods for obtaining anti-IL-6 receptor antibodies
known to those skilled in the art are not limited. Methods for substituting one or
more amino acid residues with amino acids of interest in at least one region selected
from the group consisting of the CDR, FR, and constant regions include, for example,
site-directed mutagenesis (
Hashimoto-Gotoh, T., Mizuno, T., Ogasahara, Y., and Nakagawa, M. An oligodeoxyribonucleotide-directed
dual amber method for site-directed mutagenesis. Gene (1995) 152, 271-275;
Zoller, M. J., and Smith, M. Oligonucleotide-directed mutagenesis of DNA fragments
cloned into M13 vectors. Methods Enzymol. (1983) 100, 468-500;
Kramer, W., Drutsa, V., Jansen, H. W., Kramer, B., Pflugfelder, M., and Fritz, H.
J. The gapped duplex DNA approach to oligonucleotide-directed mutation construction.
Nucleic Acids Res. (1984) 12, 9441-9456;
Kramer W., and Fritz H. J. Oligonucleotide-directed construction of mutations via
gapped duplex DNA Methods. Enzymol. (1987) 154, 350-367;
Kunkel, T. A. Rapid and efficient site-specific mutagenesis without phenotypic selection.
Proc. Natl. Acad. Sci. USA (1985) 82, 488-492). These methods can be used to substitute target amino acids in antibodies with amino
acids of interest. Methods for substituting amino acids include library technologies
such as framework shuffling (
Mol. Immunol. 2007 Apr;44(11): 3049-60) and CDR repair (
US2006/0122377). Using these methods, amino acids can be substituted into appropriate frameworks
and CDRs.
[0253] In another embodiment to obtain antibodies, an antibody that binds to IL-6 receptor
is first prepared by methods known to those skilled in the art. When the prepared
antibody is derived from a nonhuman animal, it can be humanized. Then, the prepared
antibody is tested to assess whether it has neutralizing activity by using methods
known to those skilled in the art, The binding activity and neutralizing activity
of antibodies can be determined, for example, by the methods described in the Examples;
however, such methods are not limited thereto. Next, one or more amino acid residues
in at least one selected from the group consisting of CDR, FR, and constant regions
of antibody are substituted with amino acids of interest.
[0254] More specifically, the present invention relates to methods for producing antibodies
with improved neutralizing activity, binding activity, or stability, or reduced immunogenicity,
which comprise the steps of:
- (a) expressing a DNA encoding an H chain in which one or more amino acid residues
in at least one region selected from the group consisting of CDR, FR, and constant
regions are substituted with amino acids of interest, and a DNA encoding an L chain
in which one or more amino acid residues in at least one region selected from the
group consisting of CDR and FR regions are substituted with amino acids of interest;
and
- (b) collecting the expression products of step (a).
[0255] The first step of the production methods of the present invention is expressing a
DNA encoding a mutant anti-IL-6 receptor antibody H chain in which one or more amino
acid residues in at least one region selected from the group consisting of CDR, FR,
and constant regions are substituted with amino acids of interest, and a DNA encoding
an anti-IL-6 receptor antibody L chain in which one or more amino acid residues in
at least one region selected from the group consisting of CDR and FR regions are substituted
with amino acids of interest. A DNA encoding an H chain in which one or more amino
acid residues in at least one region selected from the group consisting of CDR, FR,
and constant regions are substituted with amino acids of interest can be prepared,
for example, by obtaining a DNA encoding the CDR, FR, or constant region of a wild
type H chain, and introducing an appropriate substitution so that a codon encoding
a particular amino acid in at least one selected from the group consisting of the
CDR, FR, and constant regions encodes an amino acid of interest. Furthermore, a DNA
encoding an L chain in which one or more amino acid residues in at least one selected
from the group consisting of CDR and FR regions are substituted with amino acids of
interest can be prepared, for example, by obtaining a DNA encoding the CDR and/or
FR regions of a wild type L chain and introducing an appropriate substitution so that
a codon encoding a particular amino acid in the CDR and/or FR regions encodes an amino
acid of interest.
[0256] Alternatively, a DNA encoding an H chain in which one or more amino acid residues
in at least one selected from the group consisting of CDR, FR, and constant regions
are substituted with amino acids of interest can also be prepared by designing and
then chemically synthesizing a DNA encoding a protein in which one or more amino acid
residues in at least one selected from the group consisting of CDR, FR, and constant
regions of the wild type H chain are substituted with amino acids of interest. Furthermore,
a DNA encoding an L chain in which one or more amino acid residues in the CDR and/or
FR regions are substituted with amino acids of interest can also be prepared by designing
and then chemically synthesizing a DNA encoding a protein in which one or more amino
acid residues in the CDR and/or FR regions of a wild type L chain are substituted
with amino acids of interest.
[0257] The type of amino acid substitution includes the substitutions described herein,
but is not limited thereto.
[0258] Alternatively, a DNA encoding an H chain in which one or more amino acid residues
in at least one region selected from the group consisting of CDR, FR, and constant
regions are substituted with amino acids of interest can also be prepared as a combination
of partial DNAs. Such combinations of partial DNAs include, for example, the combination
of a DNA encoding a variable region and a DNA encoding a constant region, and the
combination of a DNA encoding an Fab region and a DNA encoding an Fc region, but are
not limited thereto. A DNA encoding an L chain can also be prepared as a combination
of partial DNAs.
[0259] Methods for expressing the above-described DNAs include the methods described below.
For example, an H chain expression vector is constructed by inserting a DNA encoding
an H chain variable region into an expression vector along with a DNA encoding an
H chain constant region. Likewise, an L chain expression vector is constructed by
inserting a DNA encoding an L chain variable region into an expression vector along
with a DNA encoding an L chain constant region. Alternatively, these H and L chain
genes may be inserted into a single vector. Expression vectors include, for example,
SV40 virus-based vectors, EB virus-based vectors, and BPV (papilloma virus)-based
vectors, but are not limited thereto.
[0260] Host cells are co-transformed with an antibody expression vector constructed by the
methods described above. Such host cells include the above-described cells such as
CHO (Chinese hamster ovary) cells as well as microorganisms such as
E. coli, yeast, and
Bacillus subtilis, and plants and animals (
Nature Biotechnology (2007) 25, 563-565;
Nature Biotechnology (1998) 16, 773-777;
Biochemical and Biophysical Research Communications (1999) 255, 444-450;
Nature Biotechnology (2005) 23, 1159-1169;
Journal of Virology (2001) 75, 2803-2809;
Biochemical and Biophysical Research Communications (2003) 308, 94-100). The transformation can be preferably achieved by using electroporation, the lipofectin
method (
R. W. Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86, 6077;
P. L. Felgner et al., Proc. Natl. Acad. Sci. USA (1987) 84, 7413), calcium phosphate method (
F. L. Graham & A. J. van der Eb, Virology (1973) 52, 456-467), DEAE-Dextran method, and the like.
[0261] In the next step of antibody production, the expression products obtained in step
(a) are collected. The expression products can be collected, for example, by culturing
the transformants and then separating the products from the transformed cells or culture
media. Separation and purification of antibodies can be achieved by an appropriate
combination of methods such as centrifugation, ammonium sulfate fractionation, salting
out, ultrafiltration, columns of 1 q, FcRn, Protein A, and Protein G, affinity chromatography,
ion exchange chromatography, and gel filtration chromatography.
[0262] Those skilled in the art can appropriately prepare the constant regions of the present
invention according to the methods for preparing antibodies.
[0263] The present invention further relates to methods for enhancing the activity of an
anti-IL-6 receptor antibody to bind or neutralize an IL-6 receptor, which comprise
at least one step selected from the group consisting of:
- (A) substituting Ser at position 1 in the amino acid sequence of SEQ ID NO: 1 (CDR1)
with another amino acid;
- (B) substituting Trp at position 5 in the amino acid sequence of SEQ ID NO: 1 (HCDR1)
with another amino acid;
- (C) substituting Tyr at position I in the amino acid sequence of SEQ ID NO: 2 (HCDR2)
with another amino acid;
- (D) substituting Thr at position 8 in the amino acid sequence of SEQ ID NO: 2 (HCDR2)
with another amino acid;
- (E) substituting Thr at position 9 in the amino acid sequence of SEQ ID NO: 2 (HCDR2)
with another amino acid;
- (F) substituting Ser at position 1 in the amino acid sequence of SEQ ID NO: 3 (HCDR3)
with another amino acid;
- (G) substituting Leu at position 2 in the amino acid sequence of SEQ ID NO: 3 (HCDR3)
with another amino acid;
- (H) substituting Thr at position 5 in the amino acid sequence of SEQ ID NO: 3 (HCDR3)
with another amino acid;
- (I) substituting Ala at position 7 in the amino acid sequence of SEQ ID NO: 3 (HCDR3)
with another amino acid;
- (J) substituting Met at position 8 in the amino acid sequence of SEQ ID NO: 3 (HCDR3)
with another amino acid;
- (K) substituting Ser at position 1 and Thr at position 5 in the amino acid sequence
of SEQ ID NO: 3 (HCDR3) with other amino acids;
- (L) substituting Leu at position 2, Ala at position 7, and Met at position 8 in the
amino acid sequence of SEQ ID NO: 3 (HCDR3) with other amino acids;
- (M) substituting Arg at position 1 in the amino acid sequence of SEQ ID NO: 4 (LCDR1)
with another amino acid;
- (N) substituting Gln at position 4 in the amino acid sequence of SEQ ID NO: 4 (LCDR1)
with another amino acid;
- (O) substituting Tyr at position 9 in the amino acid sequence of SEQ ID NO: 4 (LCDR1)
with another amino acid;
- (P) substituting Asn at position 11 in the amino acid sequence of SEQ ID NO: 4 (LCDR1)
with another amino acid;
- (Q) substituting Thr at position 2 in the amino acid sequence of SEQ ID NO: 5 (LCDR2)
with another amino acid;
- (R) substituting Gln at position 1 in the amino acid sequence of SEQ ID NO: 6 (LCDR3)
with another amino acid;
- (S) substituting Gly at position 3 in the amino acid sequence of SEQ ID NO: 6 (LCDR3)
with another amino acid;
- (T) substituting Tyr at position 9 in the amino acid sequence of SEQ ID NO: 4 (LCDR1)
and Gly at position 3 in the amino acid sequence of SEQ ID NO: 6 (LCDR3) with other
amino acids;
- (U) substituting Thr at position 5 in the amino acid sequence of SEQ ID NO: 6 (LCDR3)
with another amino acid;
- (V) substituting Gln at position 1 and Thr at position 5 in the amino acid sequence
of SEQ ID NO: 6 (LCDR3) with other amino acids; and
- (W) substituting Thr at position 9 in the amino acid sequence of SEQ ID NO: 2 (HCDR2),
and Ser at position 1 and Thr at position 5 in the amino acid sequence of SEQ ID NO:
3 (HCDR3) with other amino acids; or
- (X) a step comprising (V) and (W).
[0264] In (A) described above, the type of amino acid after substitution is not particularly
limited as long as the affinity is improved; however, substitution to Trp, Thr, Asp,
Asn, Arg, Val, Phe, Ala, Gln, Tyr, Leu, His, Glu, or Cys is preferred.
[0265] In (B) described above, the type of amino acid after substitution is not particularly
limited as long as the affinity is improved; however, substitution to Ile or Val is
preferred.
[0266] In (C) described above, the type of amino acid after substitution is not particularly
limited as long as the affinity is improved; however, substitution to Phe is preferred.
[0267] In (D) described above, the type of amino acid after substitution is not particularly
limited as long as the affinity is improved; however, substitution to Arg is preferred.
[0268] In (E) described above, the type of amino acid after substitution is not particularly
limited as long as the affinity is improved; however, substitution to Ser or Asn is
preferred.
[0269] In (F) described above, the type of amino acid after substitution is not particularly
limited as long as the affinity is improved; however, substitution to Ile, Val, Thr,
or Leu is preferred.
[0270] In (G) described above, the type of amino acid after substitution is not particularly
limited as long as the affinity is improved; however, substitution to Thr is preferred.
[0271] In (H) described above, the type of amino acid after substitution is not particularly
limited as long as the affinity is improved; however, substitution to Ala, Ile, or
Ser is preferred. Other preferred substitutions include substitution of Ser for Thr
at position 5.
[0272] In (I) described above, the type of amino acid after substitution is not particularly
limited as long as the affinity is improved; however, substitution to Ser or Val is
preferred.
[0273] In (J) described above, the type of amino acid after substitution is not particularly
limited as long as the affinity is improved; however, substitution to Leu is preferred.
[0274] In (K) described above, the type of amino acid after substitution is not particularly
limited as long as the affinity is improved; however, substitutions of Leu for Ser
at position 1 and Ala for Thr at position 5 are preferred. Other preferred substitutions
include those of Val for Ser at position 1 and Ala for Thr at position 5; Ile for
Ser at position 1 and Ala for Thr at position 5; Thr for Ser at position 1 and Ala
for Thr at position 5; Val for Ser at position 1 and Ile for Thr at position 5; Ile
for Ser at position 1 and Ile for Thr at position 5; Thr for Ser at position 1 and
Ile for Thr at position 5; and Leu for Ser at position 1 and Ile for Thr at position
5.
[0275] In (L) described above, the type of amino acid after substitution is not particularly
limited as long as the affinity is improved; however, substitution of Thr for Leu
at position 2, Val for Ala at position 7, and Leu for Met at position 8 are preferred.
[0276] In (M) described above, the type of amino acid after substitution is not particularly
limited as long as the affinity is improved; however, substitution to Phe is preferred.
[0277] In (N) described above, the type of amino acid after substitution is not particularly
limited as long as the affinity is improved; however, substitution to Arg or Thr is
preferred.
[0278] In (O) described above, the type of amino acid after substitution is not particularly
limited as long as the affinity is improved; however, substitution to Phe is preferred.
[0279] In (P) described above, the type of amino acid after substitution is not particularly
limited as long as the affinity is improved; however, substitution to Ser is preferred.
[0280] In (Q) described above, the type of amino acid after substitution is not particularly
limited as long as the affinity is improved; however, substitution to Gly is preferred.
[0281] In (R) described above, the type of amino acid after substitution is not particularly
limited as long as the affinity is improved; however, substitution to Gly, Asn, or
Ser is preferred.
[0282] In (S) described above, the type of amino acid after substitution is not particularly
limited as long as the affinity is improved; however, substitution to Ser is preferred.
[0283] In (T) described above, the type of amino acid after substitution is not particularly
limited as long as the affinity is improved; however, substitutions of Phe for Tyr
in the amino acid sequence of SEQ ID NO: 4 (LCDR1) and Ser for Gly in the amino acid
sequence of SEQ ID NO: 6 (LCDR3) are preferred.
[0284] In (U) described above, the type of amino acid after substitution is not particularly
limited as long as the affinity is improved; however, substitution to Arg or Ser is
preferred.
[0285] In (V) described above, the type of amino acid after substitution is not particularly
limited as long as the affinity is improved; however, substitutions of Gly for Gln
at position 1 and Ser for Thr at position 5 are preferred. Other preferred substitutions
include those of Gly for Gln at position 1 and Arg for Thr at position 5.
[0286] In (W) described above, substitution of Asn for Thr at position 9 in the amino acid
sequence of SEQ ID NO: 2 (HCDR2) is preferred. The preferred combinations of amino
acids after substitution for Ser at position 1 and Thr at position 5 in the amino
acid sequence of SEQ ID NO: 3 (HCDR3) include Leu and Ala, Val and Ala, Ile and Ala,
Thr and Ala, Val and Ile, Ile and Ile, Thr and Ile, and Leu and Ile.
[0287] In the steps of (A) to (X) above, the method for amino acid substitution is not particularly
limited. The substitution can be achieved, for example, by site-directed mutagenesis
described above or a method described in the Examples. When an amino acid is substituted
in a heavy chain variable region, the original amino acid sequence of the heavy chain
variable region before substitution is preferably an amino acid sequence of the heavy
chain variable region of a humanized PM-1 antibody. Alternatively, when an amino acid
is substituted in a light chain variable region, the original amino acid sequence
of the light chain variable region before substitution is preferably an amino acid
sequence of the light chain variable region of a humanized PM-1 antibody. Furthermore,
it is preferable to introduce the amino acid substitutions of steps (A) to (X) described
above into the humanized PM-1 antibody.
[0288] The methods of the present invention for enhancing the binding or neutralizing activity
of an anti-IL-6 receptor antibody comprise at least any one of the steps of (A) to
(X) described above. Specifically, the methods of the present invention may comprise
two or more of the steps of (A) to (X) described above. Furthermore, the methods of
the present invention may comprise other steps (for example, amino acid substitutions,
deletions, additions and/or insertions other than those of (A) to (X) described above)
as long as they comprise any one of the steps of (A) to (X) described above. Furthermore,
for example, FR may comprise amino acid substitutions, deletions, additions and/or
insertions, and the constant region may comprise amino acid substitutions, deletions,
additions and/or insertions. It is preferable to introduce the amino acid substitutions
described above into the humanized PM-1 antibody.
<Methods for reducing the immunogenicity risk of an anti-IL-6 receptor antibody>
[0289] The present invention also relates to methods for reducing the immunogenicity of
an anti-IL-6 receptor antibody, which comprise the step of substituting Gly for Thr
at position 2 in the amino acid sequence of SEQ ID NO: 5 (LCDR2). The methods of the
present invention for reducing the immunogenicity of an anti-IL-6 receptor antibody
may comprise other steps of amino acid substitution, as long as they comprise the
step of substituting Gly for Thr at position 2 in the amino acid sequence of SEQ ID
NO: 5 (LCDR2). The method for amino acid substitution is not particularly limited.
The substitution can be achieved, for example, by site-directed mutagenesis described
above or a method described in the Examples.
[0290] It is preferable to introduce the amino acid substitutions described above into the
humanized PM-1 antibody or a variant thereof comprising substitutions, deletions,
and/or insertions.
<Methods for lowering the isoelectric point of an anti-IL-6 receptor antibody>
[0291] The present invention also relates to methods for lowering the isoelectric point
of an anti-IL-6 receptor antibody, which comprise at least one step selected from
the group consisting of:
- (i) substituting Gln at position 16 in the amino acid sequence of SEQ ID NO: 7 (HFR1)
with another amino acid;
- (ii) substituting Arg at position 8 in the amino acid sequence of SEQ ID NO: 8 (HFR2)
with another amino acid;
- (iii) substituting Arg at position 16 in the amino acid sequence of SEQ ID NO: 9 (HFR3)
with another amino acid;
- (iv) substituting Gln at position 3 in the amino acid sequence of SEQ ID NO: 10 (HFR4)
with another amino acid;
- (v) substituting Arg at position 18 in the amino acid sequence of SEQ ID NO: 11 (LFR1)
with another amino acid;
- (vi) substituting Lys at position 11 in the amino acid sequence of SEQ ID NO: 12 (LFR2)
with another amino acid;
- (vii) substituting Gln at position 23 in the amino acid sequence of SEQ ID NO: 13
(LFR3) with another amino acid;
- (viii) substituting Lys at position 10 in the amino acid sequence of SEQ ID NO: 14
(LFR4) with another amino acid;
- (ix) substituting Ser at position 1 in the amino acid sequence of SEQ ID NO: 1 (HCDR1)
with another amino acid;
- (x) substituting Arg at position 1 in the amino acid sequence of SEQ ID NO: 4 (LCDR1)
with another amino acid;
- (xi) substituting Arg at position 4 in the amino acid sequence of SEQ ID NO: 5 (LCDR2)
with another amino acid;
- (xii) substituting Arg at position 13 in the amino acid sequence of SEQ ID NO: 7 (HFR1)
with another amino acid;
- (xiii) substituting Lys at position 15 and/or Ser at position 16 in the amino acid
sequence of SEQ ID NO: 2 (HFR1) or 100 with other amino acids;
- (xiv) substituting Gln at position 4 in the amino acid sequence of SEQ ID NO: 4 (LCDR1)
or 101 with another amino acid; and
- (xv) substituting His at position 6 in the amino acid sequence of SEQ ID NO: 5 (LCDR2)
or 103 with another amino acid.
[0292] In (i) described above, the type of amino acid after substitution is not particularly
limited as long as the isoelectric point is lowered; however, substitution to Glu
is preferred.
[0293] In (ii) described above, the type of amino acid after substitution is not particularly
limited as long as the isoelectric point is lowered; however, substitution to Glu
is preferred.
[0294] In (iii) described above, the type of amino acid after substitution is not particularly
limited as long as the isoelectric point is lowered; however, substitution to Lys
is preferred.
[0295] In (iv) described above, the type of amino acid after substitution is not particularly
limited as long as the isoelectric point is lowered; however, substitution to Glu
is preferred.
[0296] In (v) described above, the type of amino acid after substitution is not particularly
limited as long as the isoelectric point is lowered; however, substitution to Ser
is preferred.
[0297] In (vi) described above, the type of amino acid after substitution is not particularly
limited as long as the isoelectric point is lowered; however, substitution to Glu
is preferred.
[0298] In (vii) described above, the type of amino acid after substitution is not particularly
limited as long as the isoelectric point is lowered; however, substitution to Glu
is preferred.
[0299] In (viii) described above, the type of amino acid after substitution is not particularly
limited as long as the isoelectric point is lowered; however, substitution to Glu
is preferred.
[0300] In (ix) described above, the type of amino acid after substitution is not particularly
limited as long as the isoelectric point is lowered; however, substitution to Asp
is preferred.
[0301] In (x) described above, the type of amino acid after substitution is not particularly
limited as long as the isoelectric point is lowered; however, substitution to Gln
is preferred.
[0302] In (xi) described above, the type of amino acid after substitution is not particularly
limited as long as the isoelectric point is lowered; however, substitution to Glu
is preferred.
[0303] In (xii) described above, the type of amino acid after substitution is not particularly
limited as long as the isoelectric point is lowered; however, substitution to Lys
is preferred.
[0304] In (xiii) described above, the type of amino acid after substitution is not particularly
limited as long as the isoelectric point is lowered; however, substitutions to Gln
for Lys at position 15 and Asp for Ser at position 16 are preferred.
[0305] In (xiv) described above, the type of amino acid after substitution is not particularly
limited as long as the isoelectric point is lowered; however, substitution to Glu
is preferred.
[0306] In (xv) described above, the type of amino acid after substitution is not particularly
limited as long as the isoelectric point is lowered; however, substitution to Glu
is preferred.
[0307] In the steps of (i) to (xv) described above, the method for amino acid substitution
is not particularly limited. The substitution can be achieved, for example, by site-directed
mutagenesis described above or a method described in the Examples. When an amino acid
is substituted in a heavy chain variable region, the original amino acid sequence
of the heavy chain variable region before substitution is preferably an amino acid
sequence of the heavy chain variable region of a humanized PM-1 antibody. Alternatively,
when an amino acid is substituted in a light chain variable region, the original amino
acid sequence of the light chain variable region before substitution is preferably
an amino acid sequence of the light chain variable region of a humanized PM-1 antibody.
Furthermore, it is preferable to introduce the amino acid substitutions of the steps
of (i) to (xv) described above into the humanized PM-1 antibody.
[0308] The methods of the present invention for lowering the isoelectric point of an anti-IL-6
receptor antibody comprise at least any one of the steps of (i) to (xv) described
above. Specifically, the methods of the present invention may comprise two or more
of the steps of (i) to (xv) described above. Furthermore, the methods of the present
invention may comprise other steps (for example, amino acid substitutions, deletions,
additions and/or insertions other than those of (i) to (xv) described above) as long
as they comprise any one of the steps of (i) to (xv) described above. Furthermore,
for example, the constant region may comprise amino acid substitutions, deletions,
additions and/or insertions.
<Methods for improving the stability of an anti-IL-6 receptor antibody>
[0309] The present invention also relates to methods for increasing the stability of an
anti-IL-6 receptor antibody, which comprise at least one step selected from the group
consisting of:
(α) substituting Met at position 4 in the amino acid sequence of SEQ ID NO: 9 (HFR3)
with another amino acid;
(β) substituting Leu at position 5 in the amino acid sequence of SEQ ID NO: 9 (HFR3)
with another amino acid;
(γ) substituting Thr at position 9 in the amino acid sequence of SEQ ID NO: 2 (HCDR2)
with another amino acid;
(δ) substituting Thr at position 5 in the amino acid sequence of SEQ ID NO: 6 (LCDR3)
with another amino acid;
(ε) substituting Ser at position 16 in the amino acid sequence of SEQ ID NO: 2 (HCDR2)
with another amino acid; and
(ζ) substituting Ser at position 5 in the amino acid sequence of SEQ ID NO: 10 (FR4)
with another amino acid.
[0310] In (α) described above, the type of amino acid after substitution is not particularly
limited as long as the stability is improved; however, substitution to Ile is preferred.
[0311] In (β) described above, the type of amino acid after substitution is not particularly
limited as long as the stability is improved; however, substitution to Ser is preferred.
[0312] In (γ) described above, the type of amino acid after substitution is not particularly
limited as long as the stability is improved; however, substitution to Asn is preferred.
[0313] In (δ) described above, the type of amino acid after substitution is not particularly
limited as long as the stability is improved; however, substitution to Ser is preferred.
[0314] In (s) described above, the type of amino acid after substitution is not particularly
limited as long as the stability is improved; however, substitution to Gly is preferred.
[0315] In (ζ) described above, the type of amino acid after substitution is not particularly
limited as long as the stability is improved; however, substitution to Ile is preferred.
[0316] In the steps of (α) to (ζ) described above, the method for amino acid substitution
is not particularly limited. The substitution can be achieved, for example, by site-directed
mutagenesis described above or a method described in the Examples. When an amino acid
is substituted in a heavy chain variable region, the original amino acid sequence
of the heavy chain variable region before substitution is preferably an amino acid
sequence of the heavy chain variable region of a humanized PM-1 antibody. Alternatively,
when an amino acid is substituted in a light chain variable region, the original amino
acid sequence of the light chain variable region before substitution is preferably
an amino acid sequence of the light chain variable region of a humanized PM-1 antibody.
Furthermore, it is preferable to introduce the amino acid substitutions of (α) to
(ζ) described above into the humanized PM-1 antibody.
[0317] The methods of the present invention for improving the stability of an anti-IL-6
receptor antibody comprise at least any one of the steps of (α) to (ζ) described above.
Specifically, the methods of the present invention may comprise two or more of the
steps of (α) to (ζ) described above. Furthermore, the methods of the present invention
may comprise other steps (for example, amino acid substitutions, deletions, additions
and/or insertions other than those of (α) to (ζ) described above) as long as they
comprise any one of the steps of (α) to (ζ) described above. Furthermore, for example,
the constant region may comprise amino acid substitutions, deletions, additions and/or
insertions.
<Methods for reducing the immunogenicity of an anti-IL-6 receptor antibody>
[0318] The present invention also relates to methods for reducing the immunogenicity of
an anti-IL-6 receptor antibody, in particular, a humanized PM-1 antibody, which comprise
the step of substituting Lys for Arg at position 13, Glu for Gln at position 16, Ala
for Thr at position 23, and/or Ser for Thr at position 30 in the amino acid sequence
of SEQ ID NO: 7 (HFR1). The methods of the present invention for reducing the immunogenicity
of an anti-IL-6 receptor antibody may comprise other steps of amino acid substitution,
as long as they comprise the step of substituting Ser for Thr at position 30 in the
amino acid sequence of SEQ ID NO: 7 (HFR1).
[0319] The present invention further relates to methods for reducing the immunogenicity
of an anti-IL-6 receptor antibody, in particular, a humanized PM-1 antibody, which
comprise the step of substituting Val for Ala at position 27 in the amino acid sequence
of SEQ ID NO: 90 (HFR3). The methods of the present invention for reducing the immunogenicity
of an anti-IL-6 receptor antibody may comprise other steps of amino acid substitution,
as long as they comprise the step of substituting Val for Ala at position 27 in the
amino acid sequence of SEQ ID NO: 90 (HFR3).
[0320] The method for amino acid substitution is not particularly limited. The substitution
can be achieved, for example, by site-directed mutagenesis described above or a method
described in the Examples.
[0321] The present invention further relates to methods for reducing antibody immunogenicity,
which comprise converting the FR3 of an anti-IL-6 receptor antibody, in particular,
a humanized PM-1 antibody, H53/L28, or PF1 antibody, into an FR3 comprising the amino
acid sequence of SEQ ID NO: 128 or 129.
<Methods for improving antibody stability under acidic conditions>
[0322] The present invention also relates to methods for improving antibody stability under
acidic conditions, which comprise the step of substituting Met at position 276 (position
397 in the EU numbering system) in the amino acid sequence of SEQ ID NO: 20 (IgG2)
with another amino acid. The methods of the present invention for improving antibody
stability under acidic conditions may comprise other steps of amino acid substitution,
as long as they comprise the step of substituting Met at position 276 (position 397
in the EU numbering system) in the amino acid sequence of SEQ ID NO: 20 (IgG2) with
another amino acid. The type of amino acid after substitution is not particularly
limited; however, substitution to Val is preferred. The method for amino acid substitution
is not particularly limited. The substitution can be achieved, for example, by site-directed
mutagenesis described above or a method described in the Examples.
[0323] The type of target antibody is not particularly limited; however, the antibody is
preferably an anti-human IL-6 receptor antibody, more preferably a humanized PM-1
antibody or a variant thereof comprising substitutions, deletions, and/or insertions.
<Methods for reducing the heterogeneity originated from the hinge region of IgG2 constant
region>
[0324] The present invention also relates to methods for reducing antibody heterogeneity,
which comprise the step of substituting Cys at position 14 (position 131 in the EU
numbering system), Arg at position 16 (position 133 in the EU numbering system), and/or
Cys at position 102 (position 219 in the EU numbering system) in the amino acid sequence
of SEQ ID NO: 20 (IgG2) with other amino acids. The type of amino acid after substitution
is not particularly limited; however, substitutions of Ser for Cys at position 14,
Lys for Arg at position 16, and Ser for Cys at position 102 are preferred. The methods
of the present invention for reducing antibody heterogeneity may comprise other steps
of amino acid substitution, as long as they comprise the step of substituting Cys
at position 14 (position 131 in the EU numbering system), Arg at position 16 (position
133 in the EU numbering system), and/or Cys at position 102 (position 219 in the EU
numbering system) in the amino acid sequence of SEQ ID NO: 20
[0325] (IgG2). The method for amino acid substitution is not particularly limited. The substitutions
can be achieved, for example, by site-directed mutagenesis described above or a method
described in the Examples. In the amino acid substitution, all of the three amino
acids described above may be substituted or one or two (for example, positions 14
and 102) of them may be substituted.
[0326] The type of target antibody is not particularly limited; however, the antibody is
preferably an anti-human IL-6 receptor antibody, more preferably a humanized PM-1
antibody or a variant thereof comprising substitutions, deletions, and/or insertions.
<Methods for reducing the heterogeneity originated from deletion of C-terminal amino
acids in an IgG2 constant region>
[0327] The present invention also relates to methods for reducing antibody heterogeneity,
which comprise the step of deleting Gly at position 325 (position 446 in the EU numbering
system) and Lys at position 326 (position 447 in the EU numbering system) in an IgG2
constant region comprising the amino acid sequence of SEQ ID NO: 20. The methods of
the present invention for reducing antibody heterogeneity may comprise other steps
of amino acid substitution, as long as they comprise the step of deleting Gly at position
325 (position 446 in the EU numbering system) and Lys at position 326 (position 447
in the EU numbering system) in an IgG2 constant region comprising the amino acid sequence
of SEQ ID NO: 20. The method for amino acid substitution is not particularly limited.
The substitution can be achieved, for example, by site-directed mutagenesis described
above or a method described in the Examples.
[0328] The type of target antibody is not particularly limited; however, the antibody is
preferably an anti-human IL-6 receptor antibody, more preferably a humanized PM-1
antibody or a variant thereof comprising substitutions, deletions, and/or insertions.
<Methods for reducing the FcγR binding while maintaining the human sequence in the
IgG2 constant region>
[0329] The present invention also relates to methods for reducing the FcγR binding of an
antibody, which comprise the step of substituting Ser for Ala at position 209 (EU330),
Ser for Pro at position 210 (EU331), and Ala for Thr at position 218 (EU339) in an
IgG2 constant region comprising the amino acid sequence of SEQ ID NO: 20. The methods
of the present invention for reducing the FcγR binding of an antibody may comprise
other steps of amino acid substitution, as long as they comprise the step of substituting
Ser for Ala at position 209 (EU330), Ser for Pro at position 210 (EU331), and Ala
for Thr at position 218 (EU339) in an IgG2 constant region comprising the amino acid
sequence of SEQ ID NO: 20. The method for amino acid substitution is not particularly
limited. The substitution can be achieved, for example, by site-directed mutagenesis
described above or a method described in the Examples.
<Methods for improving the pharmacokinetics by substituting amino acids of IgG2 constant
regions>
[0330] The present invention also relates to methods for improving the pharmacokinetics
of an antibody, which comprise the step of substituting His at position 147 (EU268),
Arg at position 234 (EU355), and/or Gln at position 298 (EU419) in an IgG2 constant
region comprising the amino acid sequence of SEQ ID NO: 20. The methods of the present
invention for improving the pharmacokinetics of an antibody may comprise other steps
of amino acid substitution, as long as they comprise the above-described step. The
type of amino acid after substitution is not particularly limited; however, substitutions
of Gln for His at position 147 (EU268), Gln for Arg at position 234 (EU355), and Glu
for Gln at position 298 (EU419) are preferred.
[0331] The present invention also relates to methods for improving the pharmacokinetics
of an antibody, which comprise the step of substituting Asn at position 313 (EU434)
in an IgG2 constant region comprising the amino acid sequence of SEQ ID NO: 20 or
151 (M58). The type of amino acid after substitution is not particularly limited;
however, substitution to Ala is preferred. The methods of the present invention for
improving the pharmacokinetics of an antibody may comprise other steps of amino acid
substitution, as long as they comprise the above-described step.
[0332] The type of target antibody is not particularly limited; however, the antibody is
preferably an anti-human IL-6 receptor antibody, more preferably a humanized PM-1
antibody or a variant thereof comprising substitutions, deletions, and/or insertions.
[0333] The present invention also relates to methods for reducing antibody heterogeneity
originated from the hinge region of IgG2, methods for improving antibody stability
under acidic conditions, methods for reducing antibody heterogeneity originated from
C-terminus, and/or methods for reducing the FcγR binding of an antibody, all of which
comprise in an IgG2 constant region comprising the amino acid sequence of SEQ ID NO:
20 (M14ΔGK), the steps of:
- (a) substituting Ser for Ala at position 209 (position 330 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (b) substituting Ser for Pro at position 210 (position 331 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (c) substituting Ala for Thr at position 218 (position 339 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (d) substituting Val for Met at position 276 (position 397 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (e) substituting Ser for Cys at position 14 (position 131 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (f) substituting Lys for Arg at position 16 (position 133 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (g) substituting Ser for Cys at position 102 (position 219 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (h) substituting Gly for Glu at position 20 (position 137 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (i) substituting Gly for Ser at position 21 (position 138 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20; and
- (j) deleting Gly at position 325 and Lys at position 326 (positions 446 and 447 in
the EU numbering system, respectively) in the amino acid sequence of SEQ ID NO: 20.
[0334] The methods of the present invention may comprise other steps such as amino acid
substitution and deletion, as long as they comprise the steps described above. The
methods for amino acid substitution and deletion are not particularly limited. The
substitution and deletion can be achieved, for example, by site-directed mutagenesis
described above or a method described in the Examples.
[0335] The type of target antibody is not particularly limited; however, it is preferably
an anti-human IL-6 receptor antibody, more preferably a humanized PM-1 antibody or
a variant thereof comprising substitutions, deletions, and/or insertions.
[0336] The present invention also relates to methods for reducing the heterogeneity originated
from the hinge region of IgG2, methods for improving antibody stability under acidic
conditions, and/or methods for reducing antibody heterogeneity originated from C-terminus,
all of which comprise in an IgG2 constant region comprising the amino acid sequence
of SEQ ID NO: 20 (M31ΔGK), the steps of:
- (a) substituting Val for Met at position 276 (position 397 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (b) substituting Ser for Cys at position 14 (position 131 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (c) substituting Lys for Arg at position 16 (position 133 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (d) substituting Ser for Cys at position 102 (position 219 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (e) substituting Gly for Glu at position 20 (position 137 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (f) substituting Gly for Ser at position 21 (position 138 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20; and
- (g) deleting Gly at position 325 and Lys at position 326 (positions 446 and 447 in
the EU numbering system, respectively) in the amino acid sequence of SEQ ID NO: 20.
[0337] The present invention also relates to methods for reducing antibody heterogeneity
originated from the hinge region of IgG2, methods for improving antibody pharmacokinetics,
and/or methods for reducing antibody heterogeneity originated from C-terminus, all
of which comprise in an IgG2 constant region comprising the amino acid sequence of
SEQ ID NO: 20 (M58), the steps of:
- (a) substituting Ser for Cys at position 14 (position 131 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (b) substituting Lys for Arg at position 16 (position 133 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (c) substituting Ser for Cys at position 102 (position 219 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (d) substituting Gly for Glu at position 20 (position 137 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (e) substituting Gly for Ser at position 21 (position 138 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (f) substituting Gln for His at position 147 (position 268 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (g) substituting Gln for Arg at position 234 (position 355 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (h) substituting Glu for Gln at position 298 (position 419 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20; and
- (i) deleting Gly at position 325 and Lys at position 326 (positions 446 and 447 in
the EU numbering system, respectively) in the amino acid sequence of SEQ ID NO: 20.
[0338] The present invention also relates to methods for reducing antibody heterogeneity
originated from the hinge region of IgG2, methods for improving antibody pharmacokinetics,
and/or methods for reducing antibody heterogeneity originated from C-terminus, all
of which comprise in an IgG2 constant region comprising the amino acid sequence of
SEQ ID NO: 20 (M73), the steps of:
- (a) substituting Ser for Cys at position 14 (position 131 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (b) substituting Lys for Arg at position 16 (position 133 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (c) substituting Ser for Cys at position 102 (position 219 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (d) substituting Gly for Glu at position 20 (position 137 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (e) substituting Gly for Ser at position 21 (position 138 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (f) substituting Gln for His at position 147 (position 268 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (g) substituting Gln for Arg at position 234 (position 355 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (h) substituting Glu for Gln at position 298 (position 419 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20;
- (i) substituting Ala for Asn at position 313 (position 434 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 20; and
- (j) deleting Gly at position 325 and Lys at position 326 (positions 446 and 447 in
the EU numbering system, respectively) in the amino acid sequence of SEQ ID NO: 20.
[0339] The present invention also relates to methods for reducing the heterogeneity originated
from the hinge region of IgG2, methods for reducing antibody heterogeneity originated
from C-terminus, and/or methods for reducing the FcγR binding of an antibody, all
of which comprise, in an IgG2 constant region comprising the amino acid sequence of
SEQ ID NO: 20 (M86ΔGK), the steps of:
- (a) substituting Ala at position 209 (position 330 in the EU numbering system) in
the amino acid sequence of SEQ ID NO: 20 with another amino acid;
- (b) substituting Pro at position 210 (position 331 in the EU numbering system) in
the amino acid sequence of SEQ ID NO: 20 with another amino acid;
- (c) substituting Thr at position 218 (position 339 in the EU numbering system) in
the amino acid sequence of SEQ ID NO: 20 with another amino acid;
- (d) substituting Cys at position 14 (position 131 in the EU numbering system) in the
amino acid sequence of SEQ ID NO: 20 with another amino acid;
- (e) substituting Arg at position 16 (position 133 in the EU numbering system) in the
amino acid sequence of SEQ ID NO: 20 with another amino acid;
- (f) substituting Cys at position 102 (position 219 in the EU numbering system) in
the amino acid sequence of SEQ ID NO: 20 with another amino acid;
- (g) substituting Glu at position 20 (position 137 in the EU numbering system) in the
amino acid sequence of SEQ ID NO: 20 with another amino acid;
- (h) substituting Ser at position 21 (position 138 in the EU numbering system) in the
amino acid sequence of SEQ ID NO: 20 with another amino acid; and
- (i) deleting Gly at position 325 and Lys at position 326 (positions 446 and 447 in
the EU numbering system, respectively) in the amino acid sequence of SEQ ID NO: 20.
[0340] The type of amino acid after substitution is not particularly limited; however, substitutions
of Ser for Ala at position 209 (position 330 in the EU numbering system), Ser for
Pro at position 210 (position 331 in the EU numbering system), Ala for Thr at position
218 (position 339 in the EU numbering system), Ser for Cys at position 14 (position
131 in the EU numbering system), Lys for Arg at position 16 (position 133 in the EU
numbering system), Ser for Cys at position 102 (position 219 in the EU numbering system),
Gly for Glu at position 20 (position 137 in the EU numbering system), and Gly for
Ser at position 21 (position 138 in the EU numbering system) are preferred.
[0341] The present invention further relates to methods for reducing the heterogeneity originated
from the hinge region of IgG2 and/or methods for reducing antibody heterogeneity originated
from C-terminus, which comprise in an IgG2 constant region comprising the amino acid
sequence of SEQ ID NO: 20 (M40ΔGK), the steps of:
- (a) substituting Cys at position 14 (position 131 in the EU numbering system) in the
amino acid sequence of SEQ ID NO: 20 with another amino acid;
- (b) substituting Arg at position 16 (position 133 in the EU numbering system) in the
amino acid sequence of SEQ ID NO: 20 with another amino acid;
- (c) substituting Cys at position 102 (position 219 in the EU numbering system) in
the amino acid sequence of SEQ ID NO: 20 with another amino acid;
- (d) substituting Glu at position 20 (position 137 in the EU numbering system) in the
amino acid sequence of SEQ ID NO: 20 with another amino acid;
- (e) substituting Ser at position 21 (position 138 in the EU numbering system) in the
amino acid sequence of SEQ ID NO: 20 with another amino acid; and
- (f) deleting Gly at position 325 and Lys at position 326 (positions 446 and 447 in
the EU numbering system, respectively) in the amino acid sequence of SEQ ID NO: 20.
[0342] The type of amino acid after substitution is not particularly limited; however, substitutions
of Ser for Cys at position 14 (position 131 in the EU numbering system), Lys for Arg
at position 16 (position 133 in the EU numbering system), Ser for Cys at position
102 (position 219 in the EU numbering system), Gly for Glu at position 20 (position
137 in the EU numbering system), and Gly for Ser at position 21 (position 138 in the
EU numbering system) are preferred.
[0343] The methods of the present invention may comprise other steps such as amino acid
substitution and deletion, as long as they comprise the steps described above. The
methods for amino acid substitution and deletion are not particularly limited. The
substitution and deletion can be achieved, for example, by site-directed mutagenesis
described above or a method described in the Examples.
[0344] The type of target antibody is not particularly limited; however, it is preferably
an anti-human IL-6 receptor antibody, more preferably a humanized PM-1 antibody or
a variant thereof comprising substitutions, deletions, and/or insertions.
<Methods for improving the stability of an IgG4 constant region under acidic conditions>
[0345] The present invention also relates to methods for improving antibody stability under
acidic conditions, which comprise the step of substituting Arg at position 289 (position
409 in the EU numbering system) of an IgG4 constant region comprising the amino acid
sequence of SEQ ID NO: 21 (
Mol. Immunol. 1993 Jan;30(1):105-8) with another amino acid. The methods of the present invention for improving antibody
stability under acidic conditions may comprise other steps of amino acid substitution,
as long as they comprise the step of substituting Arg at position 289 (position 409
in the EU numbering system) in the amino acid sequence of SEQ ID NO: 21 (human IgG4
constant region) with another amino acid. The type of amino acid after substitution
is not particularly limited; however, substitution to Lys is preferred. The method
for amino acid substitution is not particularly limited. The substitution can be achieved,
for example, by site-directed mutagenesis described above or a method described in
the Examples.
[0346] The type of target antibody is not particularly limited; however, the antibody is
preferably an anti-human IL-6 receptor antibody, more preferably a humanized PM-1
antibody or a variant thereof comprising substitutions, deletions, and/or insertions.
<Methods for reducing the heterogeneity originated from deletion of C-terminal amino
acids in an IgG4 constant region>
[0347] The present invention also relates to methods for reducing the heterogeneity of an
antibody, which comprise the step of deleting Gly at position 326 (position 446 in
the EU numbering system) and Lys at position 327 (position 447 in the EU numbering
system) in an IgG4 constant region comprising the amino acid sequence of SEQ ID NO:
21 (
Mol. Immunol. 1993 Jan;30(1):105-8). The methods of the present invention for reducing the heterogeneity may comprise
other steps of amino acid substitution, as long as they comprise the step of deleting
Lys at position 327 (position 447 in the EU numbering system) and/or Gly at position
326 (position 446 in the EU numbering system) in an IgG4 constant region comprising
the amino acid sequence of SEQ ID NO: 21 (
Mol. Immunol. 1993 Jan;30(1):105-8). The method for amino acid substitution is not particularly limited. The substitution
can be achieved, for example, by site-directed mutagenesis described above or a method
described in the Examples.
[0348] The type of target antibody is not particularly limited; however, the antibody is
preferably an anti-human IL-6 receptor antibody, more preferably a humanized PM-1
antibody or a variant thereof comprising substitutions, deletions, and/or insertions.
[0349] The present invention also relates to methods for improving the stability under acidic
conditions, methods for reducing the heterogeneity originated from C-terminus, and/or
methods for reducing the FcγR binding of an antibody, all of which comprise in an
IgG4 constant region comprising the amino acid sequence of SEQ ID NO: 21 (
Mol. Immunol. 1993 Jan;30(1):105-8) (M11ΔGK), the steps of:
- (a) substituting Ser for Cys at position 14 (position 131 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 21;
- (b) substituting Lys for Arg at position 16 (position 133 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 21;
- (c) substituting Gly for Glu at position 20 (position 137 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 21;
- (d) substituting Gly for Ser at position 21 (position 138 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 21;
- (e) substituting Thr for Arg at position 97 (position 214 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 21;
- (f) substituting Arg for Ser at position 100 (position 217 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 21;
- (g) substituting Ser for Tyr at position 102 (position 219 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 21;
- (h) substituting Cys for Gly at position 103 (position 220 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 21;
- (i) substituting Val for Pro at position 104 (position 221 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 21;
- (j) substituting Glu for Pro at position 105 (position 222 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 21;
- (k) substituting Pro for Glu at position 113 (position 233 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 21;
- (l) substituting Val for Phe at position 114 (position 234 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 21;
- (m) substituting Ala for Leu at position 115 (position 235 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 21;
- (n) deleting Gly at position 116 (position 236 in the EU numbering system) in the
amino acid sequence of SEQ ID NO: 21;
- (o) substituting Lys for Arg at position 289 (position 409 in the EU numbering system)
in the amino acid sequence of SEQ ID NO: 21; and
- (p) deleting Gly at position 236 and Lys at position 237 (positions 446 and 447 in
the EU numbering system, respectively) in the amino acid sequence of SEQ ID NO: 21.
[0350] The methods of the present invention may comprise other steps, such as amino acid
substitution and deletion, as long as they comprise the steps described above. The
method for amino acid substitution and deletion are not particularly limited. The
substitution and deletion can be achieved, for example, by site-directed mutagenesis
described above or a method described in the Examples.
[0351] The type of target antibody is not particularly limited; however, the antibody is
preferably an anti-human IL-6 receptor antibody, more preferably a humanized PM-1
antibody or a variant thereof comprising substitutions, deletions, and/or insertions.
<Methods for reducing the heterogeneity originated from deletion of C-terminal amino
acids in an IgG1 constant region>
[0352] The present invention also relates to methods for reducing antibody heterogeneity,
which comprise the step of deleting Gly at position 329 (position 446 in the EU numbering
system) and Lys at position 330 (position 447 in the EU numbering system) in an IgG1
constant region comprising the amino acid sequence of SEQ ID NO: 19. The methods of
the present invention for reducing antibody heterogeneity may comprise other steps
of amino acid substitutions, as long as they comprise the step of deleting Lys at
position 330 (position 447 in the EU numbering system) and Gly at position 329 (position
446 in the EU numbering system) in an IgG1 constant region comprising the amino acid
sequence of SEQ ID NO: 19. The method for amino acid substitution is not particularly
limited. The substitution can be achieved, for example, by site-directed mutagenesis
described above or a method described in the Examples.
<Methods for improving the pharmacokinetics by substituting amino acids of IgG1 constant
region>
[0353] The present invention relates to methods for improving the antibody pharmacokinetics,
which comprise the step of substituting Asn at position 317 (EU434) in an IgG1 constant
region comprising the amino acid sequence of SEQ ID NO: 19 with another amino acid.
The type of amino acid after substitution is not particularly limited; however, substitution
to Ala is preferred. The methods of the present invention for improving the pharmacokinetics
may comprise other steps of amino acid substitution, as long as they comprise the
above-described step.
[0354] The present invention also relates to methods for improving the pharmacokinetics
and/or methods for reducing the heterogeneity originated from C-terminus, both of
which comprise, in an IgG1 constant region comprising the amino acid sequence of SEQ
ID NO: 19 (M83), the steps of:
- (a) substituting Ala for Asn at position 317 (EU 434) in the amino acid sequence of
(SEQ ID NO: 19; and
- (b) deleting Lys at position 330 (position 447 in the EU numbering system) and Gly
at position 329 (position 446 in the EU numbering system) in the amino acid sequence
of SEQ ID NO: 19.
[0355] The type of target antibody is not particularly limited; however, the antibody is
preferably an anti-human IL-6 receptor antibody, more preferably a humanized PM-1
antibody or a variant thereof comprising substitutions, deletions, and/or insertions.
[0356] The constant regions of the present invention described above can be combined with
any antibody variable regions, and preferably with variable regions derived from antibodies
against human IL-6 receptor. Variable regions of antibodies against human IL-6 receptor
include, for example, variable regions of a humanized PM-1 antibody. The variable
regions of a humanized PM-1 antibody may not comprise any amino acid substitutions
or may comprise substitutions such as those described above.
[0357] The present invention provides pharmaceutical compositions comprising an antibody
of the present invention. The pharmaceutical compositions of the present invention
are useful in treating diseases associated with IL-6, such as rheumatoid arthritis.
[0358] The pharmaceutical compositions of the present invention can be formulated, in addition
to the antibodies, with pharmaceutically acceptable carriers by known methods. For
example, the compositions can be used parenterally, when the antibodies are formulated
in a sterile solution or suspension for injection using water or any other pharmaceutically
acceptable liquid. For example, the compositions can be formulated by appropriately
combining the antibodies with pharmaceutically acceptable carriers or media, specifically,
sterile water or physiological saline, vegetable oils, emulsifiers, suspending agents,
surfactants, stabilizers, flavoring agents, excipients, vehicles, preservatives, binding
agents, and such, by mixing them at a unit dose and form required by generally accepted
pharmaceutical implementations. The content of the active ingredient in such a formulation
is adjusted so that an appropriate dose within the required range can be obtained.
[0359] Sterile compositions for injection can be formulated using vehicles such as distilled
water for injection, according to standard protocols.
[0360] Aqueous solutions used for injection include, for example, physiological saline and
isotonic solutions containing glucose or other adjuvants such as D-sorbitol, D-mannose,
D-mannitol, and sodium chloride. These can be used in conjunction with suitable solubilizers
such as alcohol, specifically ethanol, polyalcohols such as propylene glycol and polyethylene
glycol, and non-ionic surfactants such as Polysorbate 80
™ and HCO-50.
[0361] Oils include sesame oils and soybean oils, and can be combined with solubilizers
such as benzyl benzoate or benzyl alcohol. These may also be formulated with buffers,
for example, phosphate buffers or sodium acetate buffers; analgesics, for example,
procaine hydrochloride; stabilizers, for example, benzyl alcohol or phenol; or antioxidants.
The prepared injections are typically aliquoted into appropriate ampules.
[0362] The administration is preferably carried out parenterally, and specifically includes
injection, intranasal administration, intrapulmonary administration, and percutaneous
administration. For example, injections can be administered systemically or locally
by intravenous injection, intramuscular injection, intraperitoneal injection, or subcutaneous
injection.
[0363] Furthermore, the method of administration can be appropriately selected according
to the age and symptoms of the patient. A single dose of the pharmaceutical composition
containing an antibody or a polynucleotide encoding an antibody can be selected, for
example, from the range of 0.0001 to 1,000 mg per kg of body weight. Alternatively,
the dose may be, for example, in the range of 0.001 to 100,000 mg/person. However,
the dose is not limited to these values. The dose and method of administration vary
depending on the patient's body weight, age, and symptoms, and can be appropriately
selected by those skilled in the art.
[0364] As used herein, the three-letter and single-letter codes for respective amino acids
are as follows:
Alanine: Ala (A)
Arginine: Arg (R)
Asparagine: Asn (N)
Aspartic acid: Asp (D)
Cysteine: Cys (C)
Glutamine: Gln (Q)
Glutamic acid: Glu (E)
Glycine: Gly (G)
Histidine: His (H)
Isoleucine: Ile (I)
Leucine: Leu (L)
Lysine: Lys (K)
Methionine: Met (M)
Phenylalanine: Phe (F)
Proline: Pro (P)
Serine: Ser (S)
Threonine: Thr (T)
Tryptophan: Trp (W)
Tyrosine: Tyr (Y)
Valine: Val (V)
[0365] All prior art documents cited herein are incorporated by reference in their entirety.
Examples
[0366] Hereinbelow, the present invention is specifically described with reference to the
Examples, but it is not to be construed as being limited thereto.
[Example 1] Improvement of antigen-binding activity through CDR alteration using affinity
maturation technology
Preparation of SR344
[0368] SR344 was purified from the culture supernatant of SR344-expresssing CHO cells using
three types of column chromatography: Blue Sepharose 6 FF column chromatography, affinity
chromatography with an SR344-specific antibody-immobilized column, and gel filtration
column chromatography.
[0369] The culture supernatant was directly loaded onto a Blue Sepharose 6 FF column (GE
Healthcare Bio-Sciences) equilibrated with 20 mM Tris-HCl buffer (pH 8.0), and the
non-adsorbed fraction was thoroughly washed off using the same buffer. Then, the column
was washed with the same buffer containing 300 mM KCl. The adsorbed protein was then
eluted using the same buffer in the presence of 300 mM KCl with a linear concentration
gradient of 0 to 0.5 M KSCN. Fractions eluted with the KSCN concentration gradient
were analyzed by Western blotting using an SR344-specific antibody, and fractions
containing SR344 were collected.
[0370] The SR344-specific antibody-immobilized column was pre-equilibrated with Tris-buffered
saline (TBS). The SR344 fraction obtained in the first step was concentrated by ultrafiltration
using Amicon Ultra-15 (MILLIPORE; molecular weight cut-off of 10 kDa), and diluted
two fold with TBS before it was loaded onto the column. After the column was washed
with TBS, the adsorbed protein was eluted with 100 mM glycine-HCl buffer (pH 2.5).
The eluted fractions were neutralized by adding 1 M Tris (pH 8.1). The obtained fractions
were analyzed by SDS-PAGE to collect SR344-containing fractions.
[0371] The fraction obtained in the second step was concentrated using Amicon Ultra-15 (molecular
weight cut-off of 10 kDa) and loaded onto a Superdex 200 column (GE Healthcare Bio-Sciences)
equilibrated with PBS. The fraction eluted as the major peak was used as the final
purified sample of SR344.
Establishment of a human gp130-expressing BaF3 cell line
[0372] A BaF3 cell line expressing human gp130 was established by the procedure described
below, to obtain a cell line that proliferates in an IL-6-dependent manner.
[0373] A full-length human gp130 cDNA (
Hibi et al., Cell (1990) 63, 1149-1157 (GenBank #NM_002184)) was amplified by PCR and cloned into the expression vector
pCOS2Zeo to construct pCOS2Zeo/gp130. pCOS2Zeo is an expression vector constructed
by removing the DHFR gene expression region from pCHOI (
Hirata et al., FEBS Letter (1994) 356, 244-248) and inserting the expression region of the Zeocin resistance gene. The full-length
human IL-6R cDNA was amplified by PCR and cloned into pcDNA3.1(+) (Invitrogen) to
construct hIL-6R/pcDNA3.1(+).
[0374] 10 µg of pCOS2Zeo/gp130 was mixed with BaF3 cells (0.8 x 10
7 cells) suspended in PBS, and then pulsed at 0.33 kV and 950 µFD using Gene Pulser
(Bio-Rad). The BaF3 cells having the gene introduced by electroporation were cultured
for one whole day and night in RPMI 1640 medium (Invitrogen) supplemented with 0.2
ng/ml mouse interleukin-3 (Peprotech) and 10% Fetal Bovine Serum (hereinafter FBS;
HyClone), and selected by adding RPMI 1640 medium supplemented with 100 ng/ml human
interleukin-6 (R&D systems), 100 ng/ml human interleukin-6 soluble receptor (R&D systems),
and 10% FBS to establish a human gp130-expressing BaF3 cell line (hereinafter BaF3/gp130).
This BaF/gp130 proliferates in the presence of human interleukin-6 (R&D systems) and
SR344, and thus can be used to assess the growth inhibition activity (or IL-6 receptor
neutralizing activity) of an anti-IL-6 receptor antibody.
Construction of a library of altered CDRs
[0375] First, a humanized PM-1 antibody (
Cancer Res. 1993 Feb 15;53(4):851-6) was converted into scFv. The VH and VL regions were amplified by PCR to prepare
a humanized PM-1 HL scFv having the linker sequence GGGGSGGGGSGGGGS (SEQ ID NO: 106)
between VH and VL.
[0376] Two types of libraries were constructed by PCR using the prepared humanized PM-1
HL scFv-encoding DNA as a template. One was a target library where one of the amino
acids in a CDR is designed as X, and the other was a library where only the hot spot
sequences in a CDR are substituted with random sequences. The target library where
one of the amino acids in each CDR is designed as X was constructed as follows. The
library portion was constructed by PCR. using a primer containing NNS for the amino
acids to be incorporated into the library, while the remaining was prepared by standard
PCR. The two were linked together by assembly PCR. In this construction, only one
CDR was diversified as a library (see
J. Mol. Biol. (1996) 256, 77-88). Likewise, the library where only the hot spot sequences were substituted with random
sequences was constructed by PCR using a primer containing NNS for all hot spot amino
acids. In this construction, two libraries were constructed: one was a library where
only the hot spot in VH was diversified, and the other was a library where only the
hot spot in VL was diversified (see
Nature Biotechnology 1999 June;17:568-572).
[0377] A ribosome display library was constructed using the above-described libraries according
to
J. Immunological Methods (1999) 231, 119-135. To perform
in vitro translation based on the cell-free
E. coli system, an SDA sequence (ribosome binding site) and T7 promoter were attached to
the 5' end and a partial gene3 sequence was ligated as a ribosome display linker to
the 3' end using
SfiI.
Selection of high affinity scFv by ribosome display
[0378] Ribosome display-based panning was carried out (
Nature Biotechnology 2000 Dec:18:1287-1292). The prepared SR344 was biotinylated using NHS-PEO4-Biotin (Pierce) and then used
as an antigen. Off-rate selection was performed to obtain high affinity scFv with
high efficiency (
JBC (2004) 279(18), 18870-18877). The concentrations of biotinylated antigen and competitor antigen were 1 nM and
1 µM, respectively. The time of competition in the fourth round was 10 O/N.
scFv: insertion into phagemid, antigen binding and sequence analysis
[0379] PCR was performed to reconstruct HL scFv using the template DNA pool obtained in
the fourth round and specific primers. After digestion with
SfiI, the fragment was inserted into the phagemid vector pELBG lacI predigested with
SfiI. XL1-Blue (Stratagene) was transformed with the resulting construct. Using the yielded
colonies, antigen binding was assessed by phage ELISA and the HL scFv sequence was
analyzed. The phage ELISA was carried out using plates coated with SR344 at 1 µg/ml
(
J. Mol. Biol. (1992) 227, 381-388). Clones exhibiting SR344 binding were analyzed for their sequences using specific
primers.
Conversion of scFv into IgG, and expression and purification of IgG
[0380] IgG expression was conducted using animal cell expression vectors. Clones enriched
with a particular mutation were subjected to PCR to amplify their VLs and VHs. After
Xho/
NheI digestion and
EcoRI digestion, the amplified DNAs were inserted into an animal cell expression vector.
The nucleotide sequence of each DNA fragment was determined using a DNA sequencer
(ABI PRISM 3730xL DNA Sequencer or ABI PRISM 3700 DNA Sequencer (Applied Biosystems))
using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) according to
the method described in the attached instruction manual.
Expression of IgG-converted antibodies
[0381] Antibody expression was performed by the method described below. Human embryonic
kidney cancer-derived HEK293H cells (Invitrogen) were suspended in DMEM (Invitrogen)
supplemented with 10% Fetal Bovine Serum (Invitrogen). The cells (10-ml/plate; cell
density of 5 to 6 x 10
5 cells/ml) were plated on dishes for adherent cells (10 cm in diameter; CORNING) and
cultured in a CO
2 incubator (37°C, 5% CO
2) for one whole day and night. Then, the medium was removed by aspiration, and 6.9
ml of CHO-S-SFM-II medium (Invitrogen) was added. The prepared plasmid DNA mixture
(13.8 µg in total) was combined with 20.7 µl of 1 µg/ml Polyethylenimine (Polysciences
Inc.) and 690 µl of CHO-S-SFMII medium. The resulting mixture was incubated at room
temperature for 10 minutes, and then added to the cells in each dish. The cells were
incubated in a CO
2 incubator (at 37°C under 5% CO
2) for 4 to 5 hours. Then, 6.9 ml of CHO-S-SFM-II medium (Invitrogen) was added to
the dishes, and the cells were incubated in a CO
2 incubator for three days. The culture supernatants were collected and centrifuged
(approx. 2000 g, 5 min, room temperature) to remove the cells, and sterilized through
0.22-µm filter MILLEX
(R)-GV (Millipore). The samples were stored at 4°C until use.
Purification of IgG-converted antibodies
[0382] 50 µl of rProtein A Sepharose
™ Fast Flow (Amersham Biosciences) suspended in TBS was added to the obtained culture
supernatant, and the combined solutions were mixed by inversion at 4°C for four hours
or more. The solutions were transferred into 0.22-µm filter cups of Ultrafree
(R)-MC (Millipore). After washing three times with 500 µl of TBS, the rProtein A Sepharose
™ resin was suspended in 100 µl of 50 mM sodium acetate (pH 3.3) aqueous solution,
and the mixture was incubated for two minutes to elute the antibody. Immediately,
the eluate was neutralized by adding 6.7 µl of 1.5 M Tris-HCl (pH 7.8). Elution was
carried out twice, yielding 200 µl of purified antibody. The absorbance at 280 nm
was determined using ND-1000 Spectrophotometer (NanoDrop) or spectrophotometer DU-600
(BECKMAN) using 2 or 50 µl of the antibody solution, respectively. The antibody concentration
was calculated from the obtained value according to the following formula:

Assessment of the IgG-converted clones for human IL-6 receptor-neutralizing activity
[0383] The IL-6 receptor neutralizing activity was assessed using BaF3/gp130 which proliferates
in an IL-6/IL-6 receptor-dependent manner. After three washes with RPMI1640 supplemented
with 10% FBS, BaF31gp130 cells were suspended at 5 x 10
4 cells/ml in RPMI1640 supplemented with 60 ng/ml human interleukin-6 (TORAY), 60 ng/ml
recombinant soluble human IL-6 receptor (SR344), and 10% FBS. The cell suspensions
were dispensed (50 µl/well) into 96-well plates (CORNING). Then, the purified antibodies
were diluted with RPMI1640 containing 10% FBS, and added to each well (50 µl/well).
The cells were cultured at 37°C under 5% CO
2 for three days. WST-8 Reagent (Cell Counting Kit-8; Dojindo Laboratories) was diluted
two-fold with PBS. Immediately after 20 µl of the reagent was added to each well,
the absorbance at 450 nm (reference wavelength: 620 nm) was measured using SUNRISE
CLASSIC (TECAN). After culturing for two hours, the absorbance at 450 nm (reference
wavelength: 620 nm) was measured again. The IL-6 receptor neutralizing activity was
assessed using the change of absorbance during two hours as an indicator.
[0384] As a result, a number of antibodies whose activities were higher than that of the
humanized PM-1 antibody (wild type (WT)) were obtained. Mutations in the antibodies
whose activities were higher than that of WT are shown in Fig. 4. For example, as
shown in Fig. 1, the neutralizing activity of RD_6 was about 50 times higher than
WT in terms of 100% inhibitory concentration.
Biacore-based affinity analysis of the IgG-converted clones
[0385] The clones whose activities were higher than that of the wild type were analyzed
for antigen-antibody reaction kinetics using Biacore T100 (BIACORE). The antigen-antibody
interaction was measured by immobilizing 1800 to 2600 RU (resonance units) of rec-Protein
A (ZYMED) (hereinafter Protein A) onto a sensor chip, binding various antibodies onto
the chip, and then running the antigen over the chip as an analyte. Various concentrations
of recombinant human IL-6R sR (R&D systems) (hereinafter rhIL-6sR) were used as the
antigen. All measurements were carried out at 25°C. The kinetic parameters, association
rate constant k
a (I/Ms) and dissociation rate constant k
d (1/s) were calculated from the sensorgrams obtained by measurement. Then, K
D (M) was determined based on the rate constants. The respective parameters were determined
using Biacore T100 Evaluation Software (BIACORE).
[0386] As a result, a number of antibodies exhibiting higher affinity than the humanized
PM-1 antibody (wild type (WT)) were obtained. As an example, sensorgrams of the wild
type (WT) and RD_6 are shown in Figs. 2 and 3, respectively. The result of kinetic
parameter analysis revealed that RD_6 had about 50 times higher affinity than WT (Table
1). In addition to RD_6, antibodies exhibiting affinity dozens of times higher than
WT were also obtained. Mutations that result in higher affinity than WT are shown
in Fig. 4.
Table 1
SAMPLE |
ka (1/Ms) |
kd (1/s) |
KD (M) |
WT |
2. 8E+6 |
1. 8E-3 |
6.5E-10 |
RD_6 |
2. 3E+6 |
2. 8E-5 |
1. 2E-11 |
[Example 2] Improvement of antigen binding activity through various combinations of
CDR alterations
[0387] Mutations associated with strong activity or high affinity were combined to create
antibodies with stronger activity and higher affinity.
Production, expression, and purification of altered antibodies
[0388] Amino acids at selected sites were altered to produce altered antibodies. Specifically,
mutations were introduced into the prepared H(WT) variable region (H(WT), SEQ ID NO:
107) and L(WT) variable region (L(WT), SEQ ID NO: 108) using the QuikChange Site-Directed
Mutagenesis Kit (Stratagene) by the method described in the attached instruction manual.
After it was confirmed that the antibody H chain gene fragment inserted into a plasmid
was the humanized antibody variable region gene sequence of interest, the plasmid
was digested with
XhoI and
NotI
. A plasmid containing the antibody L chain gene fragment as an insert was digested
with
EcoRI. Then, the reaction mixtures were subjected to electrophoresis in 1% agarose gel.
A DNA fragment of the expected size (about 400 bp) was purified using the QIAquick
Gel Extraction Kit (QIAGEN) by the method described in the attached instruction manual.
The DNA was eluted with 30 µl of sterile water. Then, the antibody H chain gene fragment
was inserted into an animal cell expression vector to construct the H chain expression
vector of interest. An expression vector for the L chain was also constructed in the
same way. Ligation was carried out using the Rapid DNA Ligation Kit (Roche Diagnostics).
The
E. coli strain DH5α (Toyobo) was transformed with the plasmids. The nucleotide sequence of
each DNA fragment was determined with a DNA sequencer (ABI PRISM 3730xL DNA Sequencer
or ABI PRISM 3700 DNA Sequencer (Applied Biosystems)) using the BigDye Terminator
Cycle Sequencing Kit (Applied Biosystems) according to the method described in the
attached instruction manual. The antibodies were expressed using the constructed expression
vectors and purified by the method described in Example 1.
Assessment for the activity of neutralizing human IL-6 receptor
[0389] The purified antibodies were assessed for their neutralizing activity by the method
described in Example 1. The neutralizing activity was assessed using 600 ng/ml human
interleukin-6 (TORAY). A number of novel antibodies with stronger activity than WT
were obtained. The CDR sequences of the antibodies are shown in Fig. 5. Of them, the
antibody with the strongest activity (referred to as RDC_23) has RDC_5H as an H chain
and RDC_11L as an L chain. The neutralizing activity of RDC_23 is shown in Fig. 6.
The activity of RDC_23 was demonstrated to be about 100 times higher than WT in terms
of 100% inhibitory concentration. Improved neutralizing activity was observed not
only in RDC_23, which is an antibody having RDC_5H as an H chain and RDC_11L as an
L chain, but also in antibodies RDC_2, RDC_3, RDC_4, RDC_6, RDC_7, RDC_8, RDC_27,
RDC_28, RDC_29, RDC_30, and RDC_32, which all have L(WT) as an L chain, and RDC_2H,
RDC_3H, RDC-4H, RDC_5H, RDC_6H, RDC_7H, RDC_8H, RDC_27H, RDC_28H, RDC_29H, RDC_30H,
and RDC_32H as an H chain, respectively, as well as in an antibody referred to as
RDC_11, which has H(WT) and RDC_11L as H and L chains, respectively. It was thus shown
that antibodies having stronger neutralizing activity could be obtained by combining
mutations discovered by affinity maturation. Furthermore, since antibodies containing
such a combination of mutations had improved neutralizing activity, they were also
expected to have improved affinity.
Biacore-based affinity analysis using Protein A
[0390] Thus, of the antibodies with improved neutralizing activity, RDC_2, RDC_3, RDC_4,
RDC_5, RDC_6, RDC_7, RDC_8, RDC_11, and RDC_23 were analyzed for antigen-antibody
reaction kinetics using Biacore T100 (BIACORE). The antigen-antibody interaction was
measured by immobilizing 4400 to 5000 RU of rec-Protein A (ZYMED) immobilized onto
a sensor chip by the amine coupling method, binding various antibodies onto the chip,
and then running the antigen over the chip as an analyte. For the antigen, various
concentrations of rhIL-6sR were used. All measurements were carried out at 25°C. The
kinetic parameters, association rate constant k
a (1/Ms) and dissociation rate constant k
d (1/s) were calculated from the sensorgrams obtained by measurement. Then, K
D (M) was determined based on the rate constants. The respective parameters were determined
using Biacore T100 Evaluation Software (BIACORE). The result showed that RDC_2, RDC_3,
RDC_4, RDC_5, RDC_6, RDC_7, RDC_8, RDC_11, and RDC_23, all of which contained a combination
of mutations, had a smaller K
D value than RD_28 which contains a single mutation (Table 2). The sensorgram for RDC_23
which has a higher affinity than others is shown in Fig. 7.
Table 2
SAMPLE |
ka (1/Ms) |
kd (1/s) |
KD (M) |
RD_28 |
9.4E+05 |
1.1E-04 |
1.2E-10 |
RDC_2 |
1.1E+06 |
2.5E-05 |
2.2E-11 |
RDC_3 |
1.0E+06 |
3.7E-05 |
3.7E-11 |
RDC_4 |
1.1E+06 |
2.9E-05 |
2.7E-11 |
RDC_5 |
1.2E+06 |
2.8E-05 |
2.2E-11 |
RDC_6 |
1.2E+06 |
3.5E-05 |
2.9E-11 |
RDC_7 |
1.1E+06 |
4.2E-05 |
3.8E-11 |
RDC_8 |
1.4E+06 |
3.6E-05 |
2.5E-11 |
RDC_11 |
1.1E+06 |
7.0E-05 |
6.5E-11 |
ROC 23 |
1.2E+06 |
3.1E-05 |
2.5E-11 |
[0391] This finding suggests that these antibodies have higher affinities than the parental
antibodies that do not have the combinations of mutations. As in the case of the neutralizing
activity, this indicates that antibodies having greater affinity can be obtained by
combining mutations discovered by affinity maturation. The amino acid sequences of
variants having higher activity or affinity than WT are shown below (mutations relative
to WT are underlined).
(HCDR2)
SEQ ID NO: 45 YISYSGITNYNPSLKS
(HCDR3)
SEQ ID NO: 57 LLARATAMDY
SEQ ID NO: 58 VLARATAMDY
SEQ ID NO: 59 ILARATAMDY
SEQ ID NO: 60 TLARATAMDY
SEQ ID NO: 61 VLARITAMDY
SEQ ID NO: 62 TLARITAMDY
SEQ ID NO: 63 TLARITAMDY
SEQ ID NO: 64 LLARITAMDY
(LCDR3)
SEQ ID NO: 79 GQGNRLPYT
[0392] Specifically, an anti-IL-6 receptor antibody with markedly improved affinity and
neutralizing activity as compared to WT can be produced by designing the antibody
to have Asn at amino acid position 9 in HCDR2, Leu, Val, Ile, or Thr at amino acid
position 1 in HCDR3, Ala or Ile at amino acid position 5 in HCDR3, Gly at amino acid
position 1 in LCDR3, and Arg at amino acid position 5 in LCDR3.
Biacore-based affinity analysis using Protein A/G
[0393] WT and RDC_23 were analyzed for antigen-antibody reaction kinetics using Biacore
T100 (BIACORE). The antigen-antibody interaction was measured by immobilizing purified
Recomb Protein A/G (Pierce) (hereinafter Protein A/G) onto a sensor chip, binding
various antibodies onto the chip, and then running the antigen as an analyte over
the chip. Various concentrations of rhIL-6sR (R&D systems) and recombinant soluble
IL-6 receptor (SR344 prepared in Example 1 were used as the antigen. The sugar chain
structure of rhIL-6sR produced by baculovirus-infected insect cells is of high-mannose
type. On the other hand, the sugar chain structure of SR344 produced by CHO cells
is assumed to be of the complex sugar chain type with sialic acid at its end. Since
the sugar chain structure of soluble IL-6 receptor in an actual human body is assumed
to be of the complex sugar chain type with sialic acid at its end, SR344 is expected
to have a structure closer to that of soluble IL-6 receptor in the human body. Thus,
a comparison test between rhIL-6sR and SR344 was carried out in this experiment.
[0394] The kinetic parameters, association rate constant k
a (1/Ms) and dissociation rate constant k
d (1/s) were calculated from the sensorgrams obtained by measurement. Then, K
D (M) was determined based on the rate constants. The respective parameters were determined
using Biacore T100 Evaluation Software (BIACORE).
[0395] A sensor chip was prepared by immobilizing about 3000 RU of Protein A/G onto CM5
(BIACORE) with the amine coupling method. The kinetics of the interaction between
the two types of soluble IL-6 receptors (rhIL-6sR and SR344) and the antibodies (WT
and RDC_23) bound to Protein A/G was analyzed using the prepared sensor chip. The
running buffer used was HBS-EP+, and the flow rate was 20 µl/min. Each antibody was
prepared so that about 100 RU of the antibody was bound to Protein A/G. For the analyte,
rhIL-6sR was prepared at 0, 0.156, 0.313, and 0.625 µg/ml using HBS-EP+, while SR344
was adjusted to 0, 0.0654, 0.131, and 0.261 µg/ml. In the first step of the measurement,
the antibodies of interest, WT and RDC_23, were bound to Protein A/G, and an analyte
solution was added thereto. After three minutes of interaction, the solution was switched
with HBS-EP+ (BIACORE), and the dissociation phase was monitored for ten minutes.
After measurement of the dissociation phase, the sensor chip was regenerated by washing
with 10 µl of 10 mM glycine-HCl (pH 1.5). The association, dissociation, and regeneration
constitute one analytic cycle. All experiments were carried out at 37°C.
[0396] WT and RDC_23 were measured according to the above cycle. The resulting sensorgrams
for the two types of soluble IL-6 receptors, rhIL-6sR and SR344, are shown in Figs.
8, 9, 10, and 11. The obtained sensorgrams were kinetically analyzed using Biacore
T100 Evaluation Software, which is a data analysis software specific for Biacore (Table
3). The result showed that when comparing rhIL-6sR and SR344, the affinities of both
WT and RDC_23 for SR344 were two- to three-fold weaker For both rhIL-6sR and SR344,
RDC_23 had affinities that are about 40 to 60 times improved as compared to WT. Thus,
it was demonstrated that because of the combination of respective CDR alterations
obtained by affinity maturation, RDC_23 also had a markedly higher affinity than WT
for SR344 whose structure is presumably close to that of soluble IL-6 receptor in
the human body. All measurements described hereinafter in the Examples were carried
out at 37°C to kinetically analyze the antigen-antibody reaction using SR344 and protein
A/G.
Table 3
SAMPLE |
ANALYTE |
ka (1/Ms) |
kd (1/s) |
KD (M) |
WT |
rhIL-6sR |
1.3E+6 |
1.5E-3 |
1. 2E-9 |
SR344 |
4. 9E+5 |
2. 0E-3 |
4.0E-9 |
RDC_23 |
rhIL-6sR |
1.6E+6 |
4.5E-5 |
2.8E-11 |
SR344 |
6.4E+5 |
4. 3E-5 |
6.7E-11 |
[Example 3] Generation of H53/L28 with improved pharmacokinetics and reduced immunogenicity
risk through alterations of CDR and framework
[0397] The antibody obtained by humanizing a mouse PM-1 antibody (hereinafter referred to
as wild type or WT; the WT H and L chains are referred to as H(WT) and L(WT), respectively)
as described in
Cancer Res. 1993 Feb 15;53(4):851-6, was altered to improve the pharmacokinetics, reduce the immunogenicity risk, and
increase the stability. The alterations are described below. For the purpose of improving
the pharmacokinetics, the H and L chain variable region sequences of WT were altered
to lower the isoelectric point.
Creation of a three-dimensional structure model for the humanized PM-1 antibody
[0398] First, to identify amino acid residues exposed on the surface of the variable regions
of the humanized PM-1 antibody (H(WT)/L(WT)), a model for the Fv domain of the antibody
obtained by humanizing a mouse PM-1 antihody was created by homology modeling using
the MOE software (Chemical Computing Group Inc.).
Selection of alteration sites to reduce the isoelectric point of the humanized PM-1
antibody
[0399] A detailed analysis of the model created suggested that of the surface exposed amino
acids in the FR sequence, H16, H43, H81, H105, L18, L45, L79, and L107 (in Kabat's
numbering system; Kabat EA
et al., 1991, Sequences of Proteins of Immunological Interest, NIH), and of those in the
CDR sequence, H31, H64, H65, L24, L27, L53, and L55, were potential candidates for
the sites of alteration to reduce the isoelectric point without decreasing the activity
or stability.
Removal of remaining mouse sequences from the humanized PM-1 antibody
[0400] The humanized PM-1 antibody is an antibody whose sequence was obtained by humanizing
the mouse PM-1 antibody (
Cancer Res. 1993 Feb 15;53(4):851-6). The H chain of the humanized PM-1 antibody was obtained by grafting CDR onto the
NEW framework which is a human antibody variable region. However, mouse sequences
remain at H27, H28, H29, H30, and H71 in the H chain to maintain the activity. From
the perspective of immunogenicity risk, the best result is expected when the number
of mouse sequences is minimized. Thus, the present inventors searched for sequences
for converting H27, H28, H29, and H30 into human sequences.
Selection of alteration sites to improve the stabilily of the humanized PM-1 antibody
[0401] The present inventors speculated that it might be possible to improve the stability
of the humanized PM-1 antibody (H(WT)/L(WT)) by substituting glycine for serine at
H65 (stabilization of the turn structure; stabilization through conversion into an
HCDR2 consensus sequence), isoleucine for methionine at H69 (stabilization of the
hydrophobic core structure), serine for leucine at H70 (stabilization through replacement
of the surface exposed residue with a hydrophilic residue), asparagine for threonine
at H58 (stabilization through conversion into an HCDR2 consensus sequence), serine
for threonine at L93 (stabilization through replacement of the surface exposed residue
with a hydrophilic residue), and isoleucine for serine at H107 (stabilization of the
β sheet) in its variable regions, and considered these alterations as candidates for
increasing stability.
Removal of in silico predicted T-cell epitopes from the humanized PM-1 antibody
[0402] First, the variable regions of the humanized PM-1 antibody (H(WT)/L(WT)) were analyzed
using TEPITOPE (
Methods 2004 Dec;34(4):468-75). The result showed that the L chain CDR2 contained many T-cell epitopes that bind
to HLA. Thus, TEPITOPE analysis was carried out to find alterations that would reduce
the immunogenicity risk of the L chain CDR2 without decreasing the stability, binding
activity, or neutralizing activity. The result demonstrated that HLA-binding T-cell
epitopes can be removed without decreasing the stability, binding activity, or neutralizing
activity by substituting glycine for threonine at L51 in the L chain CDR2.
Selection of respective framework sequences
[0403] Homology search can be performed for the individual frames by using a database constructed
with the data of human antibody amino acid sequences available from the public databases:
Kabat Database (ftp://ftp.ebi.ac.uk/pub/databases/kabat/) and IMGT Database (http://imgt.cines.fr/).
From the perspectives of reducing the isoelectric point, removing remaining mouse
sequences, and improving the stability, human frameworks were selected by searching
the database for human framework sequences containing the alterations described above.
The result showed that the altered antibody H53/L28 met the requirements described
above without decreasing the binding activity or neutralizing activity when its respective
frameworks were constituted of the sequences indicated below. SOURCE indicates origins
of the human sequences. Underlined amino acid residues in each sequence represent
altered amino acids relative to WT.
[0404] Furthermore, the above-described FR3 of H53 contains a non-human sequence; thus,
it is preferable to further reduce the immunogenicity risk. A possible alteration
for reducing the immunogenicity risk is a sequence substitution resulting in an exchange
of Ala at H89 to Val (SEQ ID NO: 127). Moreover, since Arg at H71 in FR3 of H53 is
important for the binding activity (
Cancer Res. 1993 Feb 15;53(4):851-6), anti-human IL-6 receptor antibodies containing H and L chains whose frameworks
consist of a fully human sequence may be produced by using an FR3 sequence of the
human VH1 subclass (SEQ ID NO: 128) or the human VH3 subclass (SEQ ID NO: 129) where
Arg at H71 is conserved.
Selection of respective CDR sequences
[0405] The respective CDR sequences of H53/L28 were selected as shown below, from the perspectives
of reducing the isoelectric point, improving the stability, and removing T-cell epitopes,
and most importantly, not decreasing the binding activity or neutralizing activity.
Table 5
H53 |
SEQUENCE |
CDR1 |
D DHAWS |
CDR2 |
YISYSGITNYNPSLKG |
CDR3 |
SLARTTAMDY |
|
|
L28 |
SEQUENCE |
CDR1 |
QASQDISSYLN |
CDR2 |
YGSELHS |
CDR3 |
QQGNSLPYT |
Construction of expression vector for altered antibody, expression and purification
of the antibody
[0406] An expression vector for altered antibody was constructed, and the antibody was expressed
and purified by the method described in Example 1. The humanized mouse PM-1 antibody
was successively altered to have the framework and CDR sequences selected for mutagenesis
vectors for H(WT) and L(WT) of the antibody. Using the finally obtained H53/L28-encoding
animal cell expression vector (antibody amino acid sequences: H53, SEQ ID NO: 104;
and L28, SEQ ID NO: 105) having the selected framework and CDR sequences, H53/L28
was expressed and purified, and then used in the assessment described below.
Assessment of altered antibody H53/L28 for the isoelectric point by isoelectric focusing
[0407] WT and the altered antibody H53/L28 were analyzed by isoelectric focusing to assess
the change in the isoelectric point of the whole antibody caused by the amino acid
alterations in the variable regions. The procedure of isoelectric focusing is described
below. Using Phastsystem Cassette (Amersham Biosciences), Phast-Gel Dry IEF gel (Amersham
Biosciences) was rehydrated for about 30 minutes in the rehydration solution indicated
below.
Milli-Q water |
1.5 ml |
Pharmalyte 5-8 for IEF (Amersham Biosciences) |
50 µl |
Pharmalyte 8-10.5 for IEF (Amersham Biosciences) |
50 µl |
[0408] Electrophoresis was carried out in PhastSystem (Amersham Biosciences) using the rehydrated
gel according to the program indicated below. The samples were loaded onto the gel
in Step 2. Calibration Kit for pI (Amersham Biosciences) was used as the pI markers.
Step 1: |
2000 V |
2.5 mA |
3.5 W |
15°C |
75 Vh |
Step 2: |
200 V |
2.5 mA |
3.5 W |
15°C |
15 Vh |
Step 3: |
2000 V |
2.5 mA |
3.5 W |
15°C |
410 Vh |
[0409] After electrophoresis, the gel was fixed with 20% TCA, and then silver-stained using
the Silver Staining Kit, protein (Amersham Biosciences), according to the protocol
attached to the kit. After staining, the isoelectric point of the sample (the whole
antibody) was calculated from the known isoelectric points of pI markers. The result
showed that the isoelectric point of WT was about 9.3, and the isoelectric point of
the altered antibody H53/L28 was about 6.5 to 6.7. The amino acid substitution in
WT yielded H53/L28 whose isoelectric point is about 2.7 lowered. The theoretical isoelectric
point of the variable regions of H53/L28 (VH and VL sequences) was calculated by GENETYX
(GENETYX CORPORATION). The determined theoretical isoelectric point was 4.52. Meanwhile,
the theoretical isoelectric point of WT was 9.20. Thus, the amino acid substitution
in WT yielded H53/L28 having a variable region whose theoretical isoelectric point
is about 4.7 lowered.
Assessment of H53/L28 for the human IL-6 receptor-neutralizing activity
[0410] WT and H53/L28 were assessed by the method described in Example 1. The result is
shown in Fig. 12. The activity of altered antibody H53/L28 to neutralize BaF/gp130
improved several fold in comparison to WT. Specifically, the comparison of H53/L28
with WT revealed that the isoelectric point could be reduced while improving the neutralizing
activity.
Biacore-based analysis of H53/L28 for the affinity for human IL-6 receptor
[0411] The affinities of WT and H53/L28 for human IL-6 receptor were assessed by kinetic
analysis using Biacore T100 (BIACORE). The antigen-antibody interaction was measured
by immobilizing purified Recomb Protein A/G (Pierce) (hereinafter Protein A/G) onto
a sensor chip, binding various antibodies onto the chip, and then running the antigen
over the chip as an analyte. Various concentrations of recombinant soluble IL-6 receptor
(SR344) were used as the antigen. The measurement conditions were the same as described
in Example 2.
[0412] The sensorgrams obtained for WT and H53/L28 are shown in Fig. 13. Kinetic analysis
was carried out using Biacore-specific data analysis software Biacore T100 Evaluation
Software. The result is shown in Table 6. The result showed that K
D in H53/L28 was reduced about six-fold compared to WT, and this means the affinity
was improved about six-fold. Specifically, the comparison of H53/L28 with WT revealed
that the affinity could be improved six-fold while reducing the isoelectric point
at the same time. A detailed analysis suggested that the amino acid mutation that
contributed to the affinity improvement was the substitution of glycine for threonine
at L51. In other words, it is thought that the affinity can be improved by substituting
glycine for threonine at L51.
Table 6
SAMPLE |
ka (1/Ms) |
kd (1/s) |
KD (M) |
WT |
4. 9E+5 |
2. 0E-3 |
4. 0E-9 |
H53/L28 |
7. 6E+5 |
5. 2E-4 |
6. 8E-10 |
Prediction of T-cell epitopes in H53/L28 using TEPITOPE
[0413] H53/L28 was analyzed by TEPITOPE (
Methods. 2004 Dec;34(4):468-75). The result showed that the number of potential HLA-binding peptides was significantly
reduced in H53/L28 as compared to WT. This suggests reduction of the immunogenicity
risk in human.
[Example 4] Assessment of the plasma retention of H53/L28
Assessment of the altered antibody H53/L28 for its plasma pharmacokinetics in normal
mice
[0414] To assess the retention in plasma of the altered antibody H53/L28 with reduced isoelectric
point, the plasma pharmacokinetics was compared between WT and the altered antibody
H53/L28 using normal mice.
[0415] A single dose of WT or H53/L28 was intravenously or subcutaneously administered at
1 mg/kg to mice (C57BL/6J; Charles River Japan, Inc.). The blood was collected before
administration and 15 minutes, two hours, eight hours, one day, two days, five days,
seven days, 14 days, 21 days, and 28 days after administration. Note that the blood
was collected at 15 minutes after administration only from the intravenous administration
groups. The collected blood was immediately centrifuged at 4°C and 15,000 rpm for
15 minutes to obtain plasma. The separated blood plasma was stored until use in a
freezer at -20°C or below.
[0416] The concentration in the mouse plasma was determined by ELISA. First, Recombinant
Human IL-6 sR (R&D Systems) was biotinylated using EZ-LinkTM Sulfo-NFS-Biotinylation
Kit (PIERCE). The biotinylated human-sIL-6R was dispensed into Reacti-Bind Streptavidin
High Binding Capacity (HBC) Coated Plates (PIERCE), and then incubated at room temperature
for one hour or more. Thus, human-SIL-6R-immobilized plates were prepared as described
above. Mouse plasma samples and standard samples (plasma concentrations: 3.2, 1.6,
0.8, 0.4, 0.2, 0.1, and 0.05 µg/ml) were prepared and dispensed into the human-sIL-6R-immobilized
plates. The samples were incubated at room temperature for one hour, and then anti-human
IgG-AP (SIGMA) was added for reaction. After color development using the BluePhos
Microwell Phosphatase Substrates System (Kirkegaard & Perry Laboratories) as a substrate,
the absorbance at 650 nm was measured with a microplate reader. The plasma concentrations
in the mice were determined based on the absorbance of the calibration curve using
the analytical software SOFTmax PRO (Molecular Devices). The time courses for the
plasma concentrations of WT and H53/L28 after intravenous administration and subcutaneous
administration are shown in Figs. 14 and 15, respectively.
[0417] The obtained plasma concentration-time data were evaluated by a model-independent
analysis using the pharmacokinetic analysis software WinNonlin (Pharsight) to estimate
pharmacokinetics parameters (AUC, clearance (CL), and half-life (T1/2)). T1/2 was
estimated from the plasma concentrations at the last three points or those in the
terminal phase automatically selected by WinNonlin. BA was calculated from the ratio
of AUC after subcutaneous administration versus AUC after intravenous administration.
The determined pharmacokinetic parameters are shown in Table 7.
Table 7
|
iv |
|
sc |
|
|
CL |
T1/2 |
CL/F |
T1/2 |
BA |
|
mL/h/kg |
day |
mL/h/kg |
day |
% |
WT |
0.177 |
18.5 |
0.180 |
14.7 |
113 |
H53/L28 |
0.102 |
23.5 |
0.086 |
29.7 |
121 |
[0418] The half-life (T1/2) of H53/L28 in plasma after intravenous administration was prolonged
to about 1.3 times that of WT, while the clearance was reduced about 1.7 times. T1/2
of H53/L28 after subcutaneous administration was prolonged to about twice that of
WT, while the clearance was reduced about 2.1 times. Thus, the pharmacokinetics of
H53/L28 could be significantly improved by lowering the isoelectric point of WT.
[0419] H53/L28 is a humanized anti-IL-6 receptor antibody with improved binding activity
and neutralizing activity, reduced immunogenicity risk, and significantly improved
pharmacokinetics as compared to the humanized PM-1 antibody (WT). Therefore, the alterations
used to create H53/L28 may be very useful in the development of pharmaceuticals.
[Example 5] Preparation of the PF1 antibody
Construction of expression and mutagenesis vectors for the humanized PM-1 antibody
[0420] A total of four CDR mutations discovered in Example 2which improve the affinity of
RDC_23 (two each in the H and L chains) were introduced into H53/L28 created in Example
4. The H and L chains obtained by introducing the mutations of RDC_23 into H53/L28
were named PF1_H and PF1_L, respectively. The altered antibody was prepared, expressed,
and purified by the method described in Example 1. The amino acid sequences of PF1_H
and PF1_L are shown in SEQ ID NOs: 22 and 23, respectively.
Assessment for the human IL-6 receptor-neutralizing activity
[0421] The neutralizing activity of the purified PF1 antibody was assessed by the method
described in Example 1. The neutralizing activity assessment was carried out using
600 ng/ml human interleukin-6 (TORAY). The neutralizing activities of WT and PF1 are
shown in Fig. 16. PF1 was demonstrated to have an activity about 100 to 1000 times
higher than WT in terms of 100% inhibitory concentration.
Biacore-based analysis of the PF1 antibody for the affinity for human IL-6 receptor
[0422] This measurement was carried out under the same conditions described in Example 2.
The running buffer used was HBS-EP+, and the flow rate was 20 µl/min. Each antibody
was prepared so that about 100 RU of the antibody was bound to Protein A/G. SR344
was prepared at 0, 0.065, 0.131, and 0.261 µg/ml using HBS-EP+ and used as an analyte.
In the first step of the measurement, the antibody in solution was bound to Protein
A/G, and the analyte solution was allowed to interact therewith. After three minutes
of interaction, the solution was switched to HBS-EP+, and the dissociation phase was
monitored for 10 or 15 minutes. After measurement of the dissociation phase, the sensor
chip was regenerated by washing with 10 µl of 10 mM glycine-HCl (pH 1.5). The association,
dissociation, and regeneration constitute one analysis cycle. Each antibody was measured
according to this cycle.
[0423] The obtained sensorgram for PF1 is shown in Fig. 17. The sensorgram was kinetically
analyzed using the Biacore-specific data analysis software, Biacore T100 Evaluation
Software. The result is shown along with those for WT and H53/L28 in Table 8. The
result showed that the affinity of PF1 was about 150 times improved as compared to
WT. RDC_23 has a high affinity as a result of combination through affinity maturation,
and H53/L28 has an enhanced pharmacokinetics and improved affinity. Through combination
of both, PF1 obtained a higher affinity than RDC_23 or H53/L28 by an additive effect.
Table 8
SAMPLE |
ka (1/Ms) |
kd (1/s) |
KD (M) |
WT |
4.9E+05 |
2.0E-03 |
4.0E-09 |
RDC_23 |
6.4E+05 |
4.3E-05 |
6.7E-11 |
H53/L28 |
7.6E+05 |
5.2E-04 |
6.8E-10 |
PF1 |
1.3E+06 |
3.5E-05 |
2.7E-11 |
Assessment of the PF1 antibody for thermal stability by differential scanning calorimetry
(DSC)
[0424] To assess the thermal stability of the PF1 antibody, the midpoint of thermal denaturation
(Tm value) was determined by differential scanning calorimetry (DSC). The purified
antibodies of WT and PF1 were dialyzed against a solution of 20 mM sodium acetate,
150 mM NaCl, pH 6.0 (EasySEP, TOMY). DSC measurement was carried out at a heating
rate of 1 °C/min from 40 to 100°C at a protein concentration of about 0.1 mg/ml. The
result showed that the Tm of the WT Fab domain was about 94°C and that of the PF1
Fab domain was 91°C. The Tm of the Fab domain of an IgG1 type antibody molecule is
generally within the range of about 60 to 85°C (
Biochem. Biophys. Res. Commun. 2007 Apr 13;355(3):751-7;
Mol Immunol. 2007 Apr;44(11):3049-60). Thus, the observed thermal stability of the PF1 antibody was extremely high as
compared to those of typical IgG1 molecules.
Assessment of the PF1 antibody for stability at high concentrations
[0425] The PF1 antibody was assessed for stability in high concentration formulations. Purified
WT and PF1 antibodies were dialyzed against a solution of 20 mM histidine chloride,
150 mM NaCl, pH 6.5 (EasySEP, TOMY), and then concentrated by ultrafilters. The antibodies
were tested for stability at high concentrations. The conditions were as follows.
Antibodies: WT and PF1
Buffer: 20 mM histidine chloride, 150 mM NaCl, pH 6.0
Concentration: 145 mg/ml
[0426] Storage temperature and time period: 25°C for two weeks, 25°C for four weeks, or
25°C for seven weeks
[0427] Aggregation assessment method:
System: Waters Alliance
Column: G3000SWxl (TOSOH)
Mobile phase: 50 mM sodium phosphate, 300 mM KCl, pH 7.0
Flow rate, wavelength: 0.5 ml/min, 220 nm
100 times diluted samples were analyzed
[0428] The contents of aggregate in the initial formulations (immediately after preparation)
and formulations stored under various conditions were evaluated by the gel filtration
chromatography described above. Differences (amounts increased) in the content of
aggregate relative to the initial formulations are shown in Fig. 18. As a result,
the following findings were obtained: (1) both WT and PF1 were very stable; (2) the
amount of aggregate increased during seven weeks at 25°C was about 0.7% for WT and
about 0.3% for PF1, which means that the amount of aggregate increased per month at
25°C was about 0.4% and about 0.17%, respectively; and (3) PF1 was markedly stable
at high concentrations.
WO 2003/039485 has disclosed data on the stability of Daclizumab, which is available as a high concentration
IgG formulation on the market, at 25°C in a 100 mg/ml preparation. The amount of aggregate
increased per month at 25°C is about 0.3% in the formulation of 100 mg/ml Daclizumab.
Even when compared to Daclizumab, PF1 exhibits an excellent stability at high concentrations.
The increase of the number of aggregates is very problematic in developing high-concentration
liquid formulations as pharmaceuticals. The increase of PF1 antibody aggregate was
demonstrated to be very small even when the concentration of the PF1 antibody was
high.
[0429] PF1 is a molecule resulting from alteration of WT. The purposes of the alteration
include improvement of the antigen-binding activity, improvement of the pharmacokinetics
by lowering its isoelectric point, reduction of the immunogenicity risk by removing
T-cell epitopes and remaining mouse sequences, and improvement of the stability. Indeed,
the stability of PF1 in 100 mg/ml or higher concentration preparations was demonstrated
to be very high even when compared to WT. Stable and highly convenient high-concentration
formulations for subcutaneous administration can be provided by using such molecules.
[Example 6] PK/PD test of the PF1 antibody using human IL-6 receptor transgenic mice
Test for pharmacokinetics (in vivo kinetics) using human IL-6 receptor transgenic mice
[0430] WT and PF1 prepared in Example 5 were assessed for their pharmacokinetics (
in vivo kinetics) in human IL-6 receptor transgenic mice (hIL-6R tg mice;
Proc. Natl. Acad. Sci. U S A. 1995 May 23;92(11):4862-6) and their human soluble IL-6 receptor-neutralizing activity
in vivo. WT and PF1 were intravenously administered once at 10 mg/kg into hIL-6R tg mice.
Blood was collected before administration and 15 minutes, two, four, and eight hours,
one day, two, four, and seven days after administration. The blood collected was immediately
centrifuged at 4°C and 15,000 rpm for 15 minutes to obtain blood plasma. The separated
plasma was stored in a freezer at -20°C or below until use.
[0431] The concentrations in the mouse plasma were determined by ELISA. Standard samples
were prepared at 6.4, 3.2, 1.6, 0.8, 0.4, 0.2, and 0.1 µg/ml as concentrations in
plasma. Mouse plasma samples and standard samples were dispensed into immunoplates
(Nunc-Immuno Plate, MaxiSorp (Nalge nunc International)) coated with Anti-human IgG
(γ-chain specific) F(ab')2 (Sigma). The samples were incubated at room temperature
for one hour, and then Goat Anti-Human IgG-BIOT (Southern Biotechnology Associates)
and Streptavidin-alkaline phosphatase conjugate (Roche Diagnostics) were subsequently
added for reaction. After color development using the BluePhos Microwell Phosphatase
Substrates System (Kirkegaard & Perry Laboratories) as a substrate, the absorbance
at 650 nm was measured with a microplate reader. The concentrations in the mouse plasma
were determined based on the absorbance of the calibration curve using the analytical
software SOFTmax PRO (Molecular Devices). The time courses for the plasma concentrations
of WT and PF1 are shown in Fig. 19. The plasma PF1 concentration four days after administration
was about five times higher than WT. This suggests that the pharmacokinetics of PF1
of human IL-6 receptor transgenic mice is improved as compared to WT.
[0432] The human IL-6 receptor transgenic mice have been demonstrated to produce plasma
circulating human soluble IL-6 receptor. Thus, the human soluble IL-6 receptor-neutralizing
efficacy in plasma can be assessed by administering anti-human IL-6 receptor antibodies
to human IL-6 receptor transgenic mice.
[0433] The concentration of free human soluble IL-6 receptor in mouse plasma was determined
to assess the degree of neutralization of human soluble IL-6 receptor by administration
of WT or PF1. Six microliters of the mouse plasma was diluted two-fold with a dilution
buffer containing BSA. The diluted plasma was loaded onto an appropriate amount of
rProtein A Sepharose Fast Flow resin (GE Healthcare) dried in 0.22-µm filter cup (Millipore),
and all IgG type antibodies (mouse IgG, anti-human IL-6 receptor antibody, and anti-human
IL-6 receptor antibody-human soluble IL-6 receptor complex) in the plasma were adsorbed
by Protein A. Then, the solution in the cup was spinned down using a high-speed centrifuge
to collect the solution that passed through. Since the solution that passed through
does not contain Protein A-bound anti-human IL-6 receptor antibody-human soluble IL-6
receptor complex, the concentration of free soluble IL-6 receptor can be determined
by measuring the concentration of human soluble IL-6 receptor in the passed solution.
The concentration of soluble IL-6 receptor was determined using Quantikine Human IL-6
sR (R&D Systems). The concentration of free soluble IL-6 receptor in mice was measured
4, 8, 24, 48, 96, and 168 hours after administration of WT or PF1 according to the
attached instruction manual.
[0434] The result is shown in Fig. 20. In both cases of WT and PF1, the concentration of
free soluble IL-6 receptor was 10 ng/ml or less, four hours and up to eight hours
after intravenous administration of a single dose of WT or PF1 at 10 mg/kg, indicating
that the human soluble IL-6 receptor was neutralized. However, while the concentration
of free soluble IL-6 receptor was about 500 ng/ml 24 hours after WT administration,
it was 10 ng/ml or less after PF1 administration. This indicates that PF1 neutralizes
human soluble IL-6 receptor in a more sustainable way than WT.
[0435] PF1 was created by combining RDC_23 discovered through affinity maturation and H53/L28
exhibiting improved properties such as improved pharmacokinetics, and thus predicted
to be able to exhibit prolonged retention in plasma and high neutralizing activity
in vivo. Indeed, as compared to WT, PF1 was demonstrated to be more sustainable in plasma
and to exhibit a prolonged neutralizing effect in human IL-6 receptor transgenic mice
producing human soluble IL-6 receptor.
[0436] PF1 is more superior than WT (humanized PM-1 antibody) in terms of immunogenicity
risk and stability in high concentration preparations, as well as retention in plasma
and IL-6 receptor-neutralizing effect in human IL-6 receptor transgenic mice. Thus,
the alterations made to create PF1 may be very useful in the development of pharmaceuticals.
[Example 7] Improvement of the stability of IgG2 and IgG4 under acidic condition
Construction of expression vectors for IgG2- or IgG4-converted humanized IL-6 receptor
antibodies and expression of the antibodies
[0437] To reduce the Fcγ receptor binding, the constant region of humanized PM-1 antibody
(
Cancer Res. 1993 Feb 15;53(4):851-6), which is of the IgG1 isotype, was substituted with IgG2 or IgG4 (
Mol. Immunol. 1993 Jan;30(1):105-8) to generate molecules WT-IgG2 (SEQ ID NO: 109) and WT-IgG4 (SEQ ID NO: 110). An
animal cell expression vector was used to express the IgGs. An expression vector,
in which the constant region of humanized PM-1 antibody (IgG1) used in Example 1 was
digested with
NheI/
NotI and then substituted with the IgG2 or IgG4 constant region by ligation, was constructed.
The nucleotide sequence of each DNA fragment was determined with a DNA sequencer (ABI
PRISM 3730xL DNA Sequencer or ABI PRISM 3700 DNA Sequencer (Applied Biosystems)) using
the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) according to the attached
instruction manual. Using the WT L chain, WT-IgG1, WT-IgG2, and WT-IgG4 were expressed
by the method described in Example 1.
- (1) Humanized PM-1 antibody (WT-IgG1) H chain, SEQ ID NO: 15 (amino acid sequence)
- (2) WT-IgG2 H chain, SEQ ID NO: 109 (amino acid sequence)
- (3) WT-IgG4 H chain, SEQ ID NO: 110 (amino acid sequence)
Purification of WT-IgG1, WT-IgG2, and WT-IgG4 through elution from Protein A using
hydrochloric acid
[0438] 50 µl of rProtein A Sepharose
™ Fast Flow (Amersham Biosciences) suspended in TBS was added to the obtained culture
supernatant, and the combined solutions were mixed by inversion at 4°C for four hours
or more. The solutions were transferred into 0.22-µm filter cups of Ultrafree
(R)-MC (Millipore). After washing three times with 500 µl of TBS, the rProtein A Sepharose
™ resins were suspended in 100 µl of 10 mM HCI/150 mM NaCl (pH 2.0) and the mixtures
were incubated for two minutes to elute the antibodies (hydrochloric acid elution).
Immediately, the eluates were neutralized by adding 6.7 µl of 1.5 M Tris-HCl (pH 7.8).
The elution was carried out twice, yielding 200 µl of purified antibodies.
Gel filtration chromatography analysis of WT-IgG1 WT-IgG2, and WT-IgG4 purified by
hydrochloric acid elution
[0439] The contents of aggregate in the purified samples obtained by hydrochloric acid elution
were assessed by gel filtration chromatography analysis.
[0440] Aggregation assessment method:
System: Waters Alliance
Column: G3000SWxl (TOSOH)
Mobile phase: 50 mM sodium phosphate, 300 mM KCl, pH 7.0
Flow rate, wavelength: 0.5 ml/min, 220 nm
[0441] The result is shown in Fig. 21. While the content of aggregate in WT-IgG1 after purification
was about 2%, those of WT-IgG2 and WT-IgG4 after purification were about 25%. This
suggests that TgG1 is stable to acid during hydrochloric acid elution, and by contrast,
IgG2 and IgG4 are unstable and underwent denaturation/aggregation. Thus, the stability
of IgG2 and IgG4 under acidic condition was demonstrated to be lower than that of
IgG1. Protein A has been frequently used to purify IgG molecules, and the IgG molecules
are eluted from Protein A under acidic condition. In addition, virus inactivation,
which is required when developing IgG molecules as pharmaceuticals, is generally carried
out under acidic condition. It is thus desirable that the stability of IgG molecules
under acidic condition is higher. However, the stability of IgG2 and IgG4 molecules
under acidic condition was found to be lower than that of IgG1, and suggests for the
first time that there is a problem of denaturation/aggregation under acidic condition
in developing IgG2 and IgG4 molecules as pharmaceuticals. It is desirable that this
problem of denaturation/aggregation be overcome when developing them as pharmaceuticals.
To date, however, no report has been published on a method for solving this problem
through amino acid substitution.
Preparation and Assessment of WT-IgG2 and WT-IgG4 having an altered CH3 domain
[0442] The stability of IgG2 and IgG4 molecules under acidic condition was demonstrated
to be lower than that of IgG1. Thus, altered forms of IgG2 and IgG4 molecules were
tested to improve the stability under acidic condition. According to models for the
constant regions of IgG2 and IgG4 molecules, one of the potential destabilizing factors
under acidic condition was thought to be the instability at the CH3-CH3 domain interface.
As a result of various examinations, methionine at position 397 in the EU numbering
system in IgG2, or arginine at position 409 in the EU numbering system in IgG4 was
thought to destabilize the CH3/CH3 interface. Then, altered IgG2 and IgG4 antibodies
were prepared. An altered IgG2 antibody comprises the substitution of valine for methionine
at position 397 in the EU numbering system (IgG2-M397V, SEQ ID NO: 111 (amino acid
sequence)) and an altered IgG4 antibody comprises the substitution of lysine for arginine
at position 409 in the EU numbering system (IgG4-R409K, SEQ ID NO: 112 (amino acid
sequence)).
[0443] The methods used for constructing expression vectors for the antibodies of interest,
and expressing and purifying the antibodies, were the same as those used for the hydrochloric
acid elution described above. Gel filtration chromatography analysis was carried out
to estimate the contents of aggregate in the purified samples obtained by hydrochloric
acid elution from Protein A.
[0444] Aggregation assessment method:
System: Waters Alliance
Column: G3000SWxl (TOSOH)
Mobile phase: 50 mM sodium phosphate, 300 mM KCl, pH 7.0
Flow rate, wavelength: 0.5 ml/min, 220 nm
[0445] The result is shown in Fig. 21. While the content of aggregate in WT-IgG1 after purification
was about 2%, those in WT-IgG2 and WT-IgG4 after purification were about 25%. By contrast,
the contents of aggregate in variants with altered CH3 domain, IgG2-M397V and IgG4-R409K,
were comparable (approx. 2%) to that in IgG1. This finding demonstrates that the stability
of an IgG2 or IgG4 antibody under acidic condition can be improved by substituting
valine for methionine of IgG2 at position 397 in the EU numbering system or lysine
for arginine of IgG4 at position 409 in the EU numbering system, respectively. Furthermore,
the midpoint temperatures of thermal denaturation of WT-IgG2, WT-IgG4, IgG2-M397V,
and IgG4-R409K were determined by the same method as described in Example 5. The result
showed that the Tm value for the altered CH3 domain was higher in IgG2-M397V and IgG4-R409K
as compared to WT-IgG2 and WT-IgG4, respectively. This suggests that IgG2-M397V and
IgG4-R409K are also superior in terms of thermal stability as compared to WT-IgG2
and WT-IgG4, respectively.
[0446] IgG2 and IgG4 are exposed to acidic condition in virus inactivation process and in
the purification process using Protein A. Thus, denaturation/aggregation in the above
processes was problematic. However, it was discovered that the problem could be solved
by using IgG2-M397V and IgG4-R409K for the sequences of IgG2 and IgG4 constant regions.
Thus, these alterations were revealed to be very useful in developing IgG2 and IgG4
antibody pharmaceuticals. Furthermore, the usefulness of IgG2-M397V and IgG4-R409K
was also demonstrated by the finding that they are superior in thermal stability.
[Example 8] Improvement of heterogeneity derived from disulfide bonds in IgG2
Purification of WT-IgG1, WT-IgG2, and WT-IgG4 through acetic acid elution from Protein
A
[0447] 50 µl of rProtein A Sepharose
™ Fast Flow (Amersham Biosciences) suspended in TBS was added to the culture supernatants
obtained in Example 7, and the combined solutions were mixed by inversion at 4°C for
four hours or more. The solutions were transferred into 0.22-µm filter cups of Ultrafree
(R)-MC (Millipore). After washing three times with 500 µl of TBS, the rProtein A Sepharose
™ resins were suspended in 100 µl of aqueous solution of 50 mM sodium acetate (pH 3.3)
and the mixtures were incubated for two minutes to elute the antibodies. Immediately,
the eluates were neutralized by adding 6.7 µl of 1.5 M Tris-HCl (pH 7.8). The elution
was carried out twice, yielding 200 µl of purified antibodies.
Analysis of WT-IgG1, WT-IgG2 and WT-IgG4 by cation exchange chromatography (IEC)
[0448] Purified WT-IgG1, WT-IgG2, and WT-IgG4 were analyzed for homogeneity by cation exchange
chromatography.
[0449] Assessment method using IEC:
System: Waters Alliance
Column: ProPac WCX-10 (Dionex)
Mobile phase A: 25 mM MES-NaOH, pH 6.1
B: 25 mM MES-NaOH, 250 mM Na-Acetate, pH 6.1
Flow rate, wavelength: 0.5 ml/min, 280 nm
GradientB: 50%-75% (75 min) in the analysis of WT-IgG1
[0450] B: 30%-55% (75 min) in the analysis of WT-IgG2 and WT-IgG4 The result is shown in
Fig. 22. WT-IgG2 showed more than one peak in the ion exchange analysis while WT-IgG1
and WT-IgG4 exhibited a single peak. This suggests that the IgG2 molecule is more
heterogeneous as compared to IgG1 and IgG4. Indeed, IgG2 isotypes have been reported
to have heterogeneity derived from disulfide bonds in the hinge region (Non-patent
Document 30). Thus, the hetero-peaks of IgG2 shown in Fig. 22 are also assumed to
be objective substance/related substances derived from the disulfide bonds. It is
not easy to manufacture them as a pharmaceutical in large-scale while maintaining
the objective substances/related substances related heterogeneity between productions.
Thus, homogeneous (less heterogeneous) substances are desirable as much as possible
for antibody molecules to be developed as pharmaceuticals. For wild type IgG2, there
is a problem of homogeneity which is important in developing antibody pharmaceuticals.
Indeed,
US20060194280 (A1) has shown that natural IgG2 gives various hetero-peaks as a result of the disulfide
bonds in ion exchange chromatography analysis, and that the biological activity varies
among these peaks.
USA20060194280 (A1) reports refolding in the purification process as a method for combining the hetero-peaks
into a single one, but use of such a process in the production is costly and complicated.
Thus, a preferred method for combining the hetero-peaks into a single one is based
on amino acid substitution. Although the heterogeneity originated from disulfide bonds
in the hinge region should be overcome to develop IgG2 as pharmaceuticals, no report
has been published to date on a method for solving this problem through amino acid
substitution.
Preparation and assessment of altered WT-IgG2 CH1 domain and hinge region
[0451] As shown in Fig. 23, there are various potential disulfide bond patterns for an IgG2
molecule. Possible causes of the heterogeneity derived from the hinge region of IgG2
were differential pattern of disulfide bonding and free cysteines. IgG2 has two cysteines
(at positions 219 and 220 in the EU numbering system) in the upper hinge region, and
cysteines adjacent to the two upper-hinge cysteines include cysteine at position 131
in the EU numbering system in the H chain CH1 domain and L chain C-terminal cysteine,
and two corresponding cysteines in the H chain upper hinge of the dimerization partner.
Specifically, there are eight cysteines in total in the vicinity of the upper hinge
region of IgG2 when the antibody is in the associated form of H2L2. This may be the
reason for the various heterogeneous patterns due to wrong disulfide bonding and free
cysteines.
[0452] The hinge region sequence and CH1 domain of IgG2 were altered to reduce the heterogeneity
originated from the IgG2 hinge region. Examinations were conducted to avoid the heterogeneity
of IgG2 due to differential pattern of disulfide bonding and free cysteines. The result
of examining various altered antibodies suggested that the heterogeneity could be
avoided without decreasing the thermal stability by substituting serine and lysine
for cysteine and arginine at positions 131 and 133 in the EU numbering system, respectively,
in the H chain CH1 domain, and substituting serine for cysteine at position 219, EU
numbering, in the upper hinge of H chain of the wild type IgG2 constant region sequence
(hereinafter IgG2-SKSC) (IgG2-SKSC, SEQ ID NO: 120). These substitutions would enable
IgG2-SKSC to form a homogenous covalent bond between H and L chains, which is a disulfide
bond between the C-terminal cysteine of the L chain and cysteine at position 220 in
the EU numbering system (Fig. 24).
[0453] The methods described in Example 1 were used to construct an expression vector for
IgG2-SKSC and to express and purify IgG2-SKSC. The purified IgG2-SKSC and wild type
IgG2 (WT-IgG2) were analyzed for homogeneity by cation exchange chromatography. Assessment
method using IEC:
System: Waters Alliance
Column: ProPac WCX-10 (Dionex)
Mobile phase A: 25 mM MES-NaOH, pH 5.6
B: 25 mM MES-NaOH, 250 mM Na-Acetate, pH 5.6
Flow rate, wavelength: 0.5 ml/mm, 280 nm
Gradient B: 50%-100% (75 min)
[0454] The result is shown in Fig. 25. As expected above, IgG2-SKSC was shown to be eluted
at a single peak while WT-IgG2 gave multiple peaks. This suggests that the heterogeneity
derived from disulfide bonds in the hinge region of IgG2 can be avoided by using alterations
such as those used to generate IgG2-SKSC, which allow formation of a single disulfide
bond between the C-terminal cysteine of the L chain and cysteine at position 220 in
the EU numbering system. The midpoint temperatures of thermal denaturation of WT-IgG1,
WT-IgG2, and IgG2-SKSC were determined by the same methods as described in Example
5. The result showed that WT-IgG2 gave a peak for Fab domain which has a lower Tm
value than WT-IgG1, while IgG2-SKSC did not give such a peak. This suggests that IgG2-SKSC
is also superior in thermal stability as compared to WT-IgG2.
[0455] Although wild type IgG2 was thought to have a homogeneity problem which is important
in developing antibody pharmaceuticals, it was found that this problem could be solved
by using IgG2-SKSC for the constant region sequence of IgG2. Thus, IgG2-SKSC is very
useful in developing IgG2 antibody pharmaceuticals. Furthermore, the usefulness of
IgG2-SKSC was also demonstrated by the finding that it is superior in thermal stability.
[Example 9] Improvement of C-terminal heterogeneity in IgG molecules
Construction of an expression vector for H chain C-terminal ΔGK antibody from WT-IgG1
[0456] For heterogeneity of the C-terminal sequences of an antibody, deletion of C-terminal
amino acid lysine residue, and amidation of the C-terminal amino group due to deletion
of both of the two C-terminal amino acids, glycine and lysine, have been reported
(Non-patent Document 32). The absence of such heterogeneity is preferred when developing
antibody pharmaceuticals. Actually, in humanized PM-1 antibody TOCILIZUMAB, the major
component is the sequence that lacks the C-terminal amino acid lysine, which is encoded
by the nucleotide sequence but deleted in post-translational modification, and the
minor component having the lysine also coexists as heterogeneity. Thus, the C-terminal
amino acid sequence was altered to reduce the C-terminal heterogeneity. Specifically,
the present inventors altered the nucleotide sequence of wild type IgG1 to delete
the C-terminal lysine and glycine from the H chain constant region of the IgG1, and
assessed whether the amidation of the C-terminal amino group could be suppressed by
deleting the two C-terminal amino acids glycine and lysine.
[0457] Mutations were introduced into the C-terminal sequence of the H chain using pB-CH
vector encoding the humanized PM-1 antibody (WT) obtained in Example 1. The nucleotide
sequence encoding Lys at position 447 and/or Gly at position 446 in the EU numbering
system was converted into a stop codon by introducing a mutation using the QuikChange
Site-Directed Mutagenesis Kit (Stratagene) according to the method described in the
attached instruction manual. Thus, expression vectors for antibody engineered to lack
the C-terminal amino acid lysine (position 447 in the EU numbering system) and antibody
engineered to lack the two C-terminal amino acids glycine and lysine (positions 446
and 447 in the EU numbering system, respectively) were constructed. H chain C-terminal
ΔK and ΔGK antibodies were obtained by expressing the engineered H chains and the
L chain of the humanized PM-1 antibody. The antibodies were expressed and purified
by the method described in Example 1.
[0458] Purified H chain C-terminal ΔGK antibody was analyzed by cation exchange chromatography
according to the following procedure. The effect of the C-terminal deletion on heterogeneity
was assessed by cation exchange chromatography analysis using the purified H chain
C-terminal ΔGK antibody according to the method described below. The conditions of
cation exchange chromatography analysis are described below. Chromatograms for humanized
PM-1 antibody, H chain C-terminal ΔK antibody, and H chain C-terminal ΔGK antibody
were compared.
Column: ProPac WCX-10, 4 x 250 mm (Dionex)
Mobile phase A: 25 mmol/l MES/NaOH, pH 6.1
B: 25 mmol/l MES/NaOH, 250 mmol/l NaCl, pH 6.1
Flow rate: 0.5 ml/min
Gradient: 25% B (5 min) -> (105 min) -> 67% B -> (1 min) -> 100% B (5 min)
Detection: 280 nm
[0459] The analysis result for the non-altered humanized PM-1 antibody, H chain C-terminal
ΔK antibody, and H chain C-terminal ΔGK antibody is shown in Fig. 26. According to
Non-patent Document 30, a basic peak with more prolonged retention time than that
of the main peak contains an H chain C terminus with Lys at position 449 and an H
chain C terminus with amidated Pro at position 447. The intensity of the basic peak
was significantly reduced in the H chain C-terminal ΔGK antibody, while no such significant
reduction was observed in the H chain C-terminal ΔK antibody. This suggests that the
C-terminal heterogeneity of the H chain can be reduced only when the two C-terminal
amino acids are deleted from the H chain.
[0460] The temperature of thermal denaturation of the H chain C-terminal ΔGK antibody was
determined by DSC to assess the effect of the deletion of the two residues at the
H chain C terminus on thermal stability. For the DSC measurement, the antibody was
dialyzed against 20 mM acetic acid buffer (pH 6.0) containing 150 mM NaCl to change
the buffer. After thorough deaeration, the humanized PM-1 antibody and H chain C-terminal
ΔGK antibody solutions, and the reference solution (outer dialysate) were enclosed
in calorimetric cells, and thoroughly thermally equilibrated at 40°C. Then, the samples
were scanned at from 40 to 100°C with a rate of about 1K/min. The resulting denaturation
peaks were assigned (
Rodolfo et al., Immunology Letters, 1999, p 47-52). The result showed that the C-terminal deletion had no effect on the thermal denaturation
temperature of CH3 domain.
[0461] Thus, the heterogeneity originated from the C-terminal amino acid can be reduced
without affecting the thermal stability of antibody by deleting the C-terminal lysine
and glycine from the H chain constant region at the nucleotide sequence level. Since
all of the constant regions of human antibodies IgG1, IgG2, and IgG4 contain Gly and
Lys at positions 446 and 447 in the EU numbering system in their C-terminal sequences,
the method for reducing the C-terminal amino acid heterogeneity discovered in this
example and others is also expected to be applicable to IgG2 and IgG4 constant regions
and variants thereof.
[Example 10] Construction of M14ΔGK with a novel optimized constant region sequence
[0462] When an antibody pharmaceutical is aimed at neutralizing an antigen, effector functions
such as ADCC of Fc domain are unnecessary and therefore the binding to Fcγ receptor
is unnecessary. The binding to Fcγ receptor is assumed to be unfavorable from the
perspectives of immunogenicity and adverse effect (Non-patent Documents 24 and 25).
The humanized anti-IL-6 receptor IgG1 antibody TOCILIZUMAB does not need to bind to
Fcγ receptor, because it only needs to specifically bind to IL-6 receptor and neutralize
its biological activity in order to be used as a therapeutic agent for diseases associated
with IL-6, such as rheumatoid arthritis.
Construction and assessment of M14ΔGK, a Fcγ receptor-nonbinding, optimized constant
region
[0463] A possible method for impairing the Fcγ receptor binding is to convert the IgG antibody
from IgG1 isotype to IgG2 or IgG4 isotype (
Ann. Hematol. 1998 Jun;76(6):231-48). As a method for completely eliminating the binding to Fcγ receptor, a method of
introducing an artificial alteration into Fc domain has been reported. For example,
since the effector functions of anti-CD3 antibody and anti-CD4 antibody cause adverse
effects, amino acid mutations that are not present in the wild type sequence have
been introduced into the Fcγ receptor-binding region of Fc domain (Non-patent Documents
26 and 27), and the resulting Fcγ receptor-nonbinding anti-CD3 and anti-CD4 antibodies
are under clinical trials (Non-patent Documents 24 and 28). According to another report
(Patent Document 6), Fcγ receptor-nonbinding antibodies can be prepared by converting
the FcγR-binding domain of IgG1 (at positions 233, 234, 235, 236, 327, 330, and 331
in the EU numbering system) into the sequence of IgG2 (at positions 233, 234, 235,
and 236 in the EU numbering system) or IgG4 (at positions 327, 330, and 331 in the
EU numbering system). However, if all of the above mutations are introduced into IgG1,
novel peptide sequences of nine amino acids, which potentially serve as non-natural
T-cell epitope peptides, will be generated, and this increases the immunogenicity
risk. The immunogenicity risk should be minimized in developing antibody pharmaceuticals.
[0464] To overcome the above problem, alterations in the IgG2 constant region were considered.
In the FcγR-binding domain of IgG2 constant region, residues at positions 327, 330,
and 331 in the EU numbering system are different from the nonbinding sequence of IgG4
while those at positions 233, 234, 235, and 236 in the EU numbering system are amino
acids of nonbinding type. Thus, it is necessary to alter the amino acids at positions
327, 330, and 331 in the EU numbering system to the sequence of IgG4 (G2Δa described
in
Eur. J. Immunol. 1999 Aug;29(8):2613-24). However, since the amino acid at position 339 in the EU numbering system in IgG4
is alanine while the corresponding residue in IgG2 is threonine, a simple alteration
of the amino acids at positions 327, 330, and 331 in the EU numbering system to the
sequence of IgG4 unfavorably generates a novel peptide sequence of 9 amino acids,
potentially serving as a non-natural T-cell epitope peptide, and thus increases the
immunogenicity risk, which is unpreferable. Then, the present inventors found that
the generation of novel peptide sequence could be prevented by introducing the substitution
of alanine for threonine at position 339 in the EU numbering system in IgG2, in addition
to the alteration described above.
[0465] In addition to the mutations described above, other mutations were introduced, and
they were the substitution of valine for methionine at position 397 in the EU numbering
system in IgG2, which was discovered in Example 7 to improve the stability of IgG2
under acidic condition; and the substitution of serine for cysteine at position 131
in the EU numbering system, the substitution of lysine for arginine at position 133
in the EU numbering system, and the substitution of serine for cysteine at position
219 in the EU numbering system, which were discovered in Example 8 to improve the
heterogeneity originated from disulfide bonds in the hinge region. Furthermore, since
the mutations at positions 131 and 133 generate a novel peptide sequence of 9 amino
acids, potentially serving as a non-natural T-cell epitope peptide, and thus generate
the immunogenicity risk, the peptide sequence around positions 131 to 139 was converted
into a natural human sequence by introducing the substitution of glycine for glutamic
acid at position 137 in the EU numbering system and the substitution of glycine for
serine at position 138 in the EU numbering system. Furthermore, glycine and lysine
at positions 446 and 447 in the EU numbering system were deleted from the C terminus
of H chain to reduce the C-terminal heterogeneity. The constant region sequence having
all of the mutations introduced was named M14ΔGK (M14ΔGK, SEQ ID NO: 24). Although
there is a mutation of cysteine at position 219 to serine in M14ΔGK as a novel 9-amino
acid peptide sequence which potentially serves as a T-cell epitope peptide, the immunogenicity
risk was considered very low since the amino acid property of serine is similar to
that of cysteine. The immunogenicity prediction by TEPITOPE also suggested that there
was no difference in immunogenicity.
[0466] An expression vector for the antibody H chain sequence whose variable region was
WT and constant region was M14ΔGK (M14ΔGK, SEQ ID NO: 24; WT-M14ΔGK, SEQ ID NO: 113)
was constructed by the method described in Example 1. An antibody having WT-M14ΔGK
as H chain and WT as L chain was expressed and purified by the method described in
Example 1.
[0467] Furthermore, WT-M17ΔGK (M17ΔGK, SEQ ID NO: 116; WT-M17ΔGK, SEQ ID NO: 115) was constructed
with the same method by introducing mutations into the IgG1 constant region at positions
233, 234, 235, 236, 327, 330, 331, and 339 in the EU numbering system (G1Δab described
in
Eur. J. Immunol. 1999 Aug;29(8):2613-24) to impair the Fcγ receptor binding and by deleting the amino acids at positions
446 and 447 in the EU numbering system to reduce the C-terminal heterogeneity (Example
9). An expression vector for WT-M11ΔGK (M11ΔGK, SEQ ID NO: 25; WT-M11ΔGK, SEQ ID NO:
114) was constructed. In WT-M11ΔGK, mutations were introduced into the IgG4 constant
region at positions 233, 234, 235, and 236 in the EU numbering system (G4Δb described
in
Eur. J. Immunol. 1999 Aug;29(8):2613-24; this alteration newly generates non-human sequence and thus increases the immunogenicity
risk) to reduce the Fcγ receptor binding. In addition to the above alteration, to
reduce the immunogenicity risk, mutations were introduced at positions 131, 133, 137,138,
214, 217, 219, 220, 221, and 222 in the EU numbering system so that the pattern of
disulfide bonding in the hinge region was the same as that of M14ΔGK; a mutation was
introduced at position 409 in the EU numbering system (Example 7) to improve the stability
under acidic condition; and the amino acids at positions 446 and 447 in the EU numbering
system were deleted (Example 9) to reduce the C-terminal heterogeneity. WT-M17ΔGK
or WT-M11ΔGK was used as the H chain, and WT was used as the L chain. These antibodies
were expressed and purified by the method described in Example 1.
Assessment of WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK for Fcγ receptor binding
[0468] The FcγRI binding was assessed by the procedure described below. Using Biacore T100,
human-derived Fcγ receptor I (hereinafter FcγRI) immobilized onto a sensor chip was
allowed to interact with IgG1, IgG2, IgG4, M11ΔGK, M14ΔGK, or M1ΔGK 7 as an analyte.
The amounts of bound antibody were compared. The measurement was conducted using Recombinant
Human FcRIA/CD64 (R&D systems) as human-derived FcγRI, and IgG1, IgG2, IgG4, M11ΔGK,
M14ΔGK, and M17ΔGK as samples. FcγRI was immobilized onto the sensor chip CM5 (BIACORE)
by the amine coupling method. The final amount of immobilized hFcγRI was about 13000
RU. The running buffer used was HBS-EP+, and the flow rate was 20 µl/min. The sample
concentration was adjusted to 100 µg/ml using HBS-EP+. The analysis included two steps:
two minutes of association phase where 10 µl of an antibody solution was injected
and the subsequent four minutes of dissociation phase where the injection was switched
with HBS-EP+. After the dissociation phase, the sensor chip was regenerated by injecting
20 µl of 5 mM sodium hydroxide. The association, dissociation, and regeneration constitute
one analysis cycle. Various antibody solutions were injected to obtain sensorgrams.
As analytes, IgG4, IgG2, IgG1, M11, M14, and M17 were injected in this order. This
series of injection was repeated twice. The result of comparison of data on the determined
amounts of bound antibody is shown in Fig. 27. The comparison shows that the amount
of bound antibody is reduced in the order of: IgG1 > IgG4 >> IgG2 = M11ΔGK = M14ΔGK
= M17ΔGK. Thus, it was revealed that the FcγRI binding of wild type IgG2, M11ΔGK,
M14ΔGK, and M17ΔGK was weaker than that of wild type IgG1 and IgG4.
[0469] The FcγRIIa binding was assessed by the procedure described below. Using Biacore
T100, human-derived Fcγ receptor IIa (hereinafter FcγRIIa) immobilized onto a sensor
chip was allowed to interact with IgG1, IgG2, IgG4, M11ΔGK, M14ΔGK, or M17ΔGK as an
analyte. The amounts of bound antibody were compared. The measurement was conducted
using Recombinant Human FcRIIA/CD32a (R&D systems) as human-derived FcγRIIa, and IgG1,
IgG2, IgG4, M11ΔGK, M14ΔGK, and M17ΔGK as samples. FcγRIIa was immobilized onto the
sensor chip CM5 (BIACORE) by the amine coupling method. The final amount of immobilized
FcγRIIa was about 3300 RU. The running buffer used was HBS-EP+, and the flow rate
was 20 µl/min. Then, the running buffer was injected until the baseline was stabilized.
The measurement was carried out after the baseline was stabilized. The immobilized
FcγRIIa was allowed to interact with an antibody of each IgG isotype (IgG1, IgG2,
or IgG4) or antibody introduced with mutations (M11ΔGK, M14ΔGK, or M17ΔGK) as an analyte.
The amount of bound antibody was observed. The running buffer used was HBS-EP+, and
the flow rate was 20 µl/min. The measurement temperature was 25°C. The concentration
of each IgG or altered form thereof was adjusted to 100 µg/ml. 20 µl of an analyte
was injected and allowed to interact with the immobilized FcγRIIa. After interaction,
the analyte was dissociated from FcγRIIa and the sensor chip was regenerated by injecting
200 µl of the running buffer. As analytes, IgG4, IgG2, IgG1, M11ΔGK, M14ΔGK, and M17ΔGK
were injected in this order. This series of injection was repeated twice. The result
of comparison of data on the amounts of bound antibody determined is shown in Fig.
28. The comparison shows that the amount of bound antibody is reduced in the order
of: IgG1 > IgG2 = IgG4 > M11ΔGK = M14ΔGK = M17ΔGK. Thus, it was revealed that the
FcγRIIa binding of M11ΔGK, M14ΔGK, and M17ΔGK was weaker than that of wild type IgG1,
IgG2, and IgG4.
[0470] The FcγRIIb binding was assessed by the procedure described below. Using Biacore
T100, human-derived Fcγ receptor IIb (hereinafter FcγRIIb) immobilized onto a sensor
chip was allowed to interact with IgG1, IgG2, IgG4, M11ΔGK, M14ΔGK, or M17ΔGK as an
analyte. The amounts of bound antibody were compared. The measurement was conducted
using Recombinant Human FcRIIB/C (R&D systems) as human-derived FcyRIIb, and IgG1,
IgG2, IgG4, M11ΔGK, M14ΔGK, and M17ΔGK as samples. FcγRIIb was immobilized onto the
sensor chip CM5 (BIACORE) by the amine coupling method. The final amount of immobilized
FcγRIIb was about 4300 RU. Then, the running buffer was injected until the baseline
was stabilized. The measurement was carried out after the baseline was stabilized.
The immobilized FcγRIIb was allowed to interact with an antibody of each IgG isotype
(IgG1, IgG2, or IgG4) or antibody introduced with mutations (M11ΔGK, M14ΔGK, or M17ΔGK)
as an analyte. The amount of bound antibody was observed. The running buffer used
was HBS-EP+ (10 mM HEPES, 0.15 M NaCl, 3 mM EDTA, 0.05% v/v Surfactant P20), and the
flow rate was 20 µl/min. The measurement temperature was 25°C. The concentration of
each IgG or altered form thereof was adjusted to 200 µg/ml. 20 µl of an analyte was
injected and allowed to interact with the immobilized FcγRIIb. After interaction,
the analyte was dissociated from FcγRIIb and the sensor chip was regenerated by injecting
200 µl of the running buffer. As analytes, IgG4, IgG2, IgG1, M11ΔGK, M14ΔGK, and M17ΔGK
were injected in this order. This series of injection was repeated twice. The result
of comparison of data on the amounts of bound antibody determined is shown in Fig.
29. The comparison shows that the amount of bound antibody is reduced in the order
of: IgG4 > IgG1 > IgG2 > M11ΔGK = M14ΔGK = M17ΔGK. Thus, it was revealed that the
FcγRIIb binding of M11ΔGK, M14ΔGK, and M17ΔGK was weaker than that of wild type IgG1,
IgG2, and IgG4.
[0471] The FcγRIIIa binding was assessed by the procedure described below. Using Biacore
T100, human-derived Fey receptor IIIa (hereinafter FcγRIIIa) immobilized onto a sensor
chip was allowed to interact with IgG1, IgG2, IgG4, M11ΔGK, M14ΔGK, or M17ΔGK as an
analyte. The amounts of bound antibody were compared. The measurement was conducted
using hFcγRIIIaV-His6 (recombinant hFcγRIIIaV-His6 prepared in the applicants' company)
as human-derived FcγRIIIa, and IgG1, IgG2, IgG4, M11ΔGK, M14ΔGK, and M17ΔGK as samples.
FcγRIIIa was immobilized onto the sensor chip CM5 (BIACORE) by the amine coupling
method. The final amount of immobilized hFcγRIIIaV-His6 was about 8200 RU. The running
buffer used was HBS-EP+, and the flow rate was 5 µl/min. The sample concentration
was adjusted to 250 µg/ml using HBS-EP+. The analysis included two steps: two minutes
of association phase where 10 µl of an antibody solution was injected and the subsequent
four minutes of dissociation phase where the injection was switched with HBS-EP+.
After the dissociation phase, the sensor chip was regenerated by injecting 20 µl of
5 mM hydrochloric acid. The association, dissociation, and regeneration constitute
one analysis cycle. Various antibody solutions were injected to obtain sensorgrams.
As analytes, IgG4, IgG2, IgG1, M11ΔGK, M14ΔGK, and M17ΔGK were injected in this order.
The result of comparison of data on the determined amounts of bound antibody is shown
in Fig. 30. The comparison shows that the amount of bound antibody is reduced in the
order of: IgG1 >> IgG4 > IgG2 > M17ΔGK > M11ΔGK = M14ΔGK. Thus, it was revealed that
the FcγRIIIa binding of M11ΔGK, M14ΔGK, and M17ΔGK was weaker than that of wild type
IgG1, IgG2, and IgG4. Furthermore, the FcγRIIIa binding of M11ΔGK and M14ΔGK was found
to be weaker than that of M17ΔGK containing the mutation G1Δab reported in
Eur. J. Immunol. 1999 Aug;29(8):2613-24.
[0472] The finding described above demonstrates that the Fcγ receptor binding of WT-M14ΔGK,
WT-M17ΔGK, and WT-M11ΔGK is markedly reduced as compared to wild type IgG1. The immunogenicity
risk due to Fcγ receptor-mediated internalization into APC and adverse effects caused
by the effector function such as ADCC can be avoided by using WT-M14ΔGK, WT-M17ΔGK,
or WT-M11ΔGK as a constant region. Thus, WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK are useful
as constant region sequence of antibody pharmaceuticals aimed at neutralizing antigens.
Assessment of WT-M14ΔGK WT-M17ΔGK and WT-M11ΔGK for stability at high concentrations
[0473] WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK were assessed for stability at high concentrations.
The purified antibodies of WT-IgG1, WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK were dialyzed
against a solution of 20 mM histidine chloride, 154 mM NaCl, pH 6.5 (EasySEP, TOMY),
and then concentrated by ultrafilters. The antibodies were tested for stability at
high concentrations. The conditions were as follows.
Antibodies: WT-IgG1, WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK
Buffer: 20 mM histidine chloride, 150 mM NaCl, pH 6.5
Concentration: 61 mg/ml
[0474] Storage temperature and time period: 40°C for two weeks, 40°C for one month, 40°C
for two months
[0475] Aggregation assessment method:
System: Waters Alliance
Column: G3000SWxl (TOSOH)
Mobile phase: 50 mM sodium phosphate, 300 mM KCl, pH 7.0
Flow rate, wavelength: 0.5 ml/mm, 220 nm
100 times diluted samples were analyzed
[0476] The contents of aggregate in the initial formulations (immediately after preparation)
and formulations stored under various conditions were estimated by gel filtration
chromatography described above. Differences (amounts increased) in the content of
aggregate relative to the initial formulations are shown in Fig. 31. The result showed
that the amounts of aggregate in WT-M14ΔGK, WT-M17ΔGK, and WT-M11ΔGK increased only
slightly as compared to WT-IgG1 and were about half of the content in WT. Furthermore,
as shown in Fig. 32, the amount of increased Fab fragment was comparable between WT-IgG1
and WT-M17ΔGK, while the amounts increased in WT-M14ΔGK and WT-M11ΔGK were about one
quarter of the amount in WT. Degeneration pathways of IgG type antibody formulations
include formation of aggregate and generation of Fab degradate as described in
WO 2003/039485. Based on the two criteria, aggregation and Fab fragment generation, WT-M14ΔGK and
WT-M11ΔGK were demonstrated to have a superior stability in formulations as compared
to WT-IgG1. Thus, even for antibodies that have an IgG1 constant region with poor
stability and could not be prepared as antibody pharmaceuticals in high-concentration
liquid formulation, the use of WT-M14ΔGK, WT-M17ΔGK, or WT-M11ΔGK as a constant region
was expected to allow production of more stable high-concentration liquid formulations.
[0477] In particular, M14ΔGK was expected to be very useful as a novel constant region sequence
that would (1) overcome the instability of the original IgG2 molecule under acidic
condition; (2) improve the heterogeneity originated from disulfide bonds in the hinge
region; (3) not bind to Fcγ receptor; (4) have a minimized number of novel peptide
sequences of 9 amino acids which potentially serve as T-cell epitope peptides; and
(5) have a better stability than IgG1 in high-concentration formulations.
[Example 11] Preparation of PF1-M14ΔGK antibody
[0478] The variable region of PF1 (whose constant region is IgG1) constructed in Example
5 was excised using
XhoI and
NheI. The constant region of M14ΔGK (whose variable region is WT) constructed in Example
7 was excised using
NheI and
NotI. The two antibody H chain gene fragments were inserted into an animal cell expression
vector to construct an expression vector for the H chain of interest, PF1-M14ΔGK (PF1_H-M14ΔGK,
SEQ ID NO: 117). The L chain used was PF1_L. The antibody PF1-M14ΔGK was expressed
and purified by the method described in Example 1.
[0479] The antibody PF1-M14ΔGK was superior in various aspects as compared to WT (humanized
PM-1 antibody) and thus expected to be very useful as anti-IL-6 receptor antibody
pharmaceuticals.
[Example 12] Preparation and assessment of M31ΔGK
[0480] M14ΔGK prepared in Example 10 was altered by substituting the IgG2 sequence for the
amino acids at positions 330, 331, and 339 in the EU numbering system to construct
M31ΔGK (M31ΔGK, SEQ ID NO: 118). An expression vector for a sequence of antibody H
chain whose variable region is WT and constant region sequence is M31ΔGK (WT-M31ΔGK,
SEQ ID NO: 119) was constructed by the method described in Example 1. Using WT-M31ΔGK
H chain and WT L chain, WT-M31 was expressed and purified by the method described
in Example 1.
[0481] In addition to WT-M31, WT-IgG2 and WT-M14ΔGK were expressed and purified at the same
time, and analyzed by cation exchange chromatography by the procedure described below.
The conditions used in the cation exchange chromatography analysis were as follows.
Chromatograms for WT-IgG2, WT-M14ΔGK, and WT-M31ΔGK were compared.
Column: ProPac WCX-10,4 x 250 mm (Dionex)
Mobile phase A: 25 mmol/l MES/NaOH, pH 6.1
B: 25 mmol/l MES/NaOH, 250 mmol/l NaCl, pH 6.1
Flow rate: 0.5 ml/min
Gradient: 0% B (5 min) -> (65 min) -> 100% B -> (1 min)
Detection: 280 nm
[0482] The analysis result for WT-IgG2, WT-M14ΔGK, and WT-M31ΔGK is shown in Fig. 33. Like
WT-M14ΔGK, WT-M31ΔGK was demonstrated to be eluted as a single peak, while WT-IgG2
gave multiple peaks. This indicates that the heterogeneity derived from disulfide
bonds in the hinge region of IgG2 can also be avoided in WT-M31ΔGK.
[Example 13] Preparation of a fully humanized antibody F2H/L39-IgG1
Full humanization of the framework sequence of the PF1 antibody
[0483] Arginine at position 71 (Kabat's numbering system; Kabat EA
et al. 1991. Sequences of Proteins of Immunological Interest. NIH) is the only mouse sequence
that remains in PF1_H prepared in Example 5. This is unpreferable from the perspective
of immunogenicity. In general, the residue at position 71 in the H chain is an important
sequence for the conformation of HCDR2. In fact, it has been reported that during
generation of the humanized PM1 antibody, the residue at position 71 is essential
for the binding activity of mouse PM1 antibody. The binding activity was demonstrated
to be significantly reduced by substituting valine at position 71 (
Cancer Research 53, 851-856, 1993). Meanwhile, PF1_H is classified into the VH4 family of human germ line genes, and
valine at position 71 is highly conserved in the VH4 family. The neutralizing activity
was also demonstrated to be significantly reduced by substituting valine for arginine
at position 71.
[0484] Thus, to completely remove the mouse sequence while maintaining the arginine at position
71, the present inventors searched among sequences of human germ line genes and reported
human antibodies for sequences that have arginine at position 71 and share conserved
residues important for the maintenance of antibody tertiary structure. As a result,
the inventors discovered a candidate sequence which contains important conserved residues
although its homology to PF1_H is low as shown in Table 9.

[0485] H96-IgG1 (amino acid sequence of SEQ ID NO: 134) was designed by substituting the
above-described candidate sequence for the region of positions 66 to 94 in PF1_H-IgG1,
Kabat's numbering. The antibody variable region was prepared by PCR (assembly PCR)
using a combination of synthetic oligo-DNAs. The constant region was amplified from
an expression vector for IgG1 by PCR. The antibody variable region and constant region
were linked together by assembly PCR, and then inserted into an animal cell expression
vector. H96/PF1L-IgG1 was expressed and purified by the method described in Example
1.
Assessment of H96/PF1L-IgG1, an antibody with fully humanized framework
[0486] The Tm of purified H96/PF1L-IgG1 was determined by the method described in Example
5. The affinity measurement was carried out under essentially the same conditions
used in Example 5. Note that the concentration of SR344 was adjusted to 0, 0.36, and
1.4 µg/ml, and the dissociation phase was monitored for 15 minutes. The result showed
that the Tm and affinity of H96/PF1L-IgG1 were almost the same as those of PF1-IgG1
(Table 10).
Table 10
|
Tm (°C) |
ka(1/Ms) |
kd (1/s) |
KD(M) |
PF1 ANTIBODY |
91.3 |
1. 4E+06 |
4.2E-05 |
3.1E-11 |
H96/PF1L-IgG1 |
89.8 |
1.2E+06 |
4.8E-05 |
3.9E-11 |
[0487] As described above, the present inventors generated an antibody with a fully humanized
PF1 antibody framework, using H96 for the PF1 antibody H chain to completely remove
the remaining mouse sequence from the PF1 antibody while maintaining its Tm and affinity.
Since the framework sequence of H96/PF1L-IgG1 has no mouse-derived sequence, H96/PF1L-IgG1
is expected to be superior, especially from the perspective of immunogenicity.
Construction of F2H/L39-IgG1 with lowered pI and attenuated immunogenicity risk
[0488] As demonstrated in Example 4, the pharmacokinetics can be enhanced by lowering pI
through alteration of amino acids in the antibody variable region. Thus, the amino
acid substitutions shown below were further introduced into H96-IgG1 constructed above.
To lower pI, glutamine was substituted for lysine at position 64, and aspartic acid
was substituted for glycine at position 65. Furthermore, to reduce the immunogenicity
risk, glutamine was substituted for glutamic acid at position 105 and isoleucine was
substituted for threonine at position 107. In addition, to achieve affinity enhancement
such as that in Example 2, alternation was introduced where leucine was substituted
for valine at position 95 and alanine was substituted for isoleucine at position 99.
To prepare F2H-IgG1 (amino acid sequence of SEQ ID NO: 135), these amino acid substitutions
were introduced into H96-IgG1 by the method described in Example 1.
[0489] Furthermore, the following amino acid substitutions were introduced into PF1L. To
lower pI, glutamic acid was substituted for glutamine at position 27 and glutamic
acid was substituted for leucine at position 55. To prepare L39 (amino acid sequence
of SEQ ID NO: 136), these amino acid substitutions were introduced into PF1L by the
method described in Example 1. Using F2H-IgG1 as heavy chain and L39 as light chain,
F2H/L39-IgG1 was expressed and purified by the method described in Example 1.
Biacore-based analysis of F2H/L39-IgG1 for the affinity for human IL-6 receptor
[0490] Humanized PM1 antibody (wild type (WT)), PF1 antibody (constructed in Example 5),
and F2H/L39-IgG1 were analyzed for affinity. This measurement was carried out under
essentially the same conditions used in Example 4. Note that the concentration of
SR344 was adjusted to 0, 0.36, and 1.4 µg/ml, and the dissociation phase was monitored
for 15 minutes (Table 11).
Table 11
SAMPLE |
ka(1/Ms) |
kd(1/s) |
KD (M) |
PF1-IgG1 |
1.5E+06 |
4.4E-05 |
3.0E-11 |
F2H/L39-IgG1 |
7.7E+05 |
4.0E-05 |
5.2E-11 |
[0491] The result showed that F2H/L39-IgG1 had very strong affinity (maintaining a KD in
the order of 10
-11) but its k
a was decreased to about half of that of PF1-IgG1.
Assessment of F2H/L39-IgG1 for its human IL-6 receptor-neutralizing activity
[0492] The neutralizing activities of humanized PM1 antibody (wild type (WT)) and F2H/L39-IgG1
were assessed by the method described in Example 1. The assessment of neutralizing
activity was carried out using 600 ng/ml human interleukin-6 (TORAY). As shown in
Fig. 34, F2H/L39-IgG1 was demonstrated to have a very strong activity, 100 or more
times higher than WT in terms of 100% inhibitory concentration.
Assessment of F2H/L39-IgG1 for its isoelectric point by isoelectric focusing
[0493] The isoelectric point of F2H/L39-IgG1 was determined by the method described in Example
3. The isoelectric point of F2H/L39-IgG1 was 5.5, suggesting that its pharmacokinetics
was improved due to a lower isoelectric point relative to the PF1 antibody prepared
in Example 5.
[0494] The theoretical isoelectric point of the variable regions of F2H/L39 (VH and VL sequences)
was calculated to be 4.3 by using GENETYX (GENETYX CORPORATION). Meanwhile, the theoretical
isoelectric point of WT was 9.20. Thus, WT has been converted through amino acid substitution
into F2H/L39 which has a variable region with a theoretical isoelectric point decreased
by about 4.9.
PK/PD test of F2H/L39-IgG1 using cynomolgus monkeys
[0495] The humanized PM1 antibody (wild type (WT)), PF1 antibody, and F2H/L39-IgG1 were
assessed for their pharmacokinetics (PK) and pharmacodynamics (PD) in cynomolgus monkeys.
WT, PF1, and F2H/L39-IgG1 were subcutaneously administered once at 1.0 mg/kg, and
blood was collected before administration and over the time course. The concentration
of each antibody in plasma was determined in the same way as described in Example
6. The plasma concentration time courses of WT, PF1, and F2H/L39-IgG1 are shown in
Fig. 35. The efficacy of each antibody to neutralize membrane-bound cynomolgus monkey
IL-6 receptor was assessed. Cynomolgus monkey IL-6 was administered subcutaneously
in the lower back at 5 µg/kg every day from Day 3 to Day 10 after antibody administration,
and the CRP concentration in each animal was determined 24 hours later. The time courses
of CRP concentration after administration of WT or F2H/L39 are shown in Fig. 36. To
assess the efficacy of each antibody to neutralize soluble cynomolgus monkey IL-6
receptor, the concentration of free soluble cynomolgus monkey IL-6 receptor in the
plasma of cynomolgus monkeys was determined. The time courses of free soluble cynomolgus
monkey IL-6 receptor concentration after administration of WT or F2H/L39 are shown
in Fig. 37.
[0496] These results showed that the plasma concentration time courses of WT and PF 1 were
comparable to each other; however, the plasma concentration of F2H/L39-IgG1, which
has a lowered pI, was maintained higher than that of these two antibodies. Meanwhile,
when compared to WT, F2H/L39-IgG1 which has a high affinity for IL-6 receptor was
found to maintain lower concentrations of CRP and free soluble cynomolgus monkey IL-6
receptor.
[Example 14] Assessment of the plasma retention of WT-M14
Method for estimating the retention in human plasma
[0497] The prolonged retention (slow elimination) of IgG molecule in plasma is known to
be due to the function of FcRn which is known as a salvage receptor of IgG molecule
(
Nat. Rev. Immunol. 2007 Sep;7(9):715-25). When taken up into endosomes via pinocytosis, under the acidic conditions within
endosome (approx. pH 6.0), IgG molecules bind to FcRn expressed in endosomes. While
IgG molecules that do not bind to FcRn are transferred and degraded in lysosomes,
those bound to FcRn are translocated to the cell surface and then released from FcRn
back into plasma again under the neutral conditions in plasma (approx. pH 7.4).
[0498] Known IgG-type antibodies include the IgG1, IgG2, IgG3, and IgG4 isotypes. The plasma
half-lives of these isotypes in human are reported to be about 36 days for IgG1 and
IgG2; about 29 days for IgG3; and 16 days for IgG4 (
Nat. Biotechnol. 2007 Dec; 25(12):1369-72). Thus, the retention of IgG1 and IgG2 in plasma is believed to be the longest. In
general, the isotypes of antibodies used as pharmaceutical agents are IgG1, IgG2,
and IgG4. Methods reported for further improving the pharmacokinetics of these IgG
antibodies include methods for improving the above-described binding to human FcRn,
and this is achieved by altering the sequence of IgG constant region (
J. Biol. Chem. 2007 Jan 19;282(3):1709-17;
J. Immunol. 2006 Jan 1;176(1):346-56).
Assessment of the binding to human FcRn
[0500] FcRn is a complex of FcRn and β2-microglobulin. Oligo-DNA primers were prepared based
on the human FcRn gene sequence disclosed (
J. Exp. Med. (1994) 180 (6), 2377-2381). A DNA fragment encoding the whole gene was prepared by PCR using human cDNA (Human
Placenta Marathon-Ready cDNA, Clontech) as a template and the prepared primers. Using
the obtained DNA fragment as a template, a DNA fragment encoding the extracellular
domain containing the signal region (Met1-Leu290) was amplified by PCR, and inserted
into an animal cell expression vector (the amino acid sequence of human FcRn as set
forth in SEQ ID NO: 140). Likewise, oligo-DNA primers were prepared based on the human
β2-microglobulin gene sequence disclosed (
Proc. Natl. Acad. Sci. USA. (2002) 99 (26), 16899-16903). A DNA fragment encoding the whole gene was prepared by PCR using human cDNA (Hu-Placenta
Marathon-Ready cDNA, CLONTECH) as a template and the prepared primers. Using the obtained
DNA fragment as a template, a DNA fragment encoding the whole β2-microglobulin containing
the signal region (Met1-Met119) was amplified by PCR and inserted into an animal cell
expression vector (the amino acid sequence of human β2-microglobulin as set forth
in SEQ ID NO: 141).
[0501] Soluble human FcRn was expressed by the following procedure. The plasmids constructed
for human FcRn and β2-microglobulin were introduced into cells of the human embryonic
kidney cancer-derived cell line HEK293H (Invitrogen) using 10% Fetal Bovine Serum
(Invitrogen) by lipofection. The resulting culture supernatant was collected, and
FcRn was purified using IgG Sepharose 6 Fast Flow (Amersham Biosciences) by the method
described in
J. Immunol. 2002 Nov 1;169(9):5171-80, followed by further purification using HiTrap Q HP (GE Healthcare).
[0502] The binding to human FcRn was assessed using Biacore 3000. An antibody was bound
to Protein L or rabbit anti-human IgG Kappa chain antibody immobilized onto a sensor
chip, human FcRn was added as an analyte for interaction with the antibody, and the
affinity (KD) was calculated from the amount of bound human FcRn. Specifically, Protein
L or rabbit anti-human IgG Kappa chain antibody was immobilized onto sensor chip CM5
(BIACORE) by the amine coupling method using 50 mM Na-phosphate buffer (pH 6.0) containing
150 mM NaCl as the running buffer. Then, an antibody was diluted with a running buffer
containing 0.02% Tween20, and injected to be bound to the chip. Human FcRn was then
injected and the binding of the human FcRn to antibody was assessed.
[0503] The affinity was computed using BIAevaluation Software. The obtained sensorgram was
used to calculate the amount of hFcRn bound to the antibody immediately before the
end of human FcRn injection. The affinity of the antibody for human FcRn was calculated
by fitting with the steady state affinity method.
Assessment for the plasma retention in human FcRn transgenic mice
[0504] The pharmacokinetics in human FcRn transgenic mice (B6.mFcRn-/-.hFcRn Tg line 276
+/+ mice; Jackson Laboratories) was assessed by the following procedure. An antibody
was intravenously administered once at a dose of 1 mg/kg to mice, and blood was collected
at appropriate time points. The collected blood was immediately centrifuged at 15,000
rpm and 4°C for 15 minutes to obtain blood plasma. The separated plasma was stored
in a freezer at -20°C or below until use. The plasma concentration was determined
by ELISA.
Predictive assessment of the plasma retention of WT-M14 in human
[0505] The bindings of WT-IgG1 and WT-M14 to bind to human FcRn were assessed by BIAcore.
As shown in Table 12, the result indicated that the binding of WT-M14 was slightly
greater than that of WT-IgG1.
Table 12
|
KD(µM) |
WT-IgG1 |
2.07 |
WT-M14 |
1.85 |
[0506] As shown in Fig. 38, however, the retention in plasma was comparable between WT-IgG1
and WT-M14 when assessed using human FcRn transgenic mice. This finding suggests that
the plasma retention of the M14 constant region in human is comparable to that of
the IgG1 constant region.
[Example 15] Preparation of WT-M44, WT-M58, and WT-M73 which have improved pharmacokinetics
Preparation of the WT-M58 molecule
[0507] As described in Example 14, the plasma retention of WT-M14 in human FcRn transgenic
mice was comparable to that of WT-IgG1. Known methods to improve pharmacokinetics
include those to lower the isoelectric point of an antibody and those to enhance the
binding to FcRn. Here, the modifications described below were introduced to improve
the pharmacokinetics of WT-M14. Specifically, the following substitutions were introduced
into WT-M31ΔGK, which was prepared from WT-M 14 as described in Example 4: substitution
of methionine for valine at position 397; substitution of glutamine for histidine
at position 268; substitution of glutamine for arginine at position 355; and substitution
of glutamic acid for glutamine at position 419 in the EU numbering system. These four
substitutions were introduced into WT-M31ΔGK to generate WT-M58 (amino acid sequence
of SEQ ID NO: 142). Expression vectors were prepared by the same method described
in Example 1. WT-M58 and L(WT) were used as H chain and L chain, respectively. WT-M58
was expressed and purified by the method described in Example 1.
Construction of the WT-M73 molecule
[0508] On the other hand, WT-M44 (amino acid sequence of SEQ ID NO: 143) was generated by
introducing into IgG1 a substitution of alanine for the amino acid at position 434,
EU numbering. WT-M83 (amino acid sequence of SEQ ID NO: 185) was also generated by
deletions of glycine at position 446, EU numbering and lysine at position 447, EU
numbering to reduce H chain C-terminal heterogeneity. Furthermore, WT-M73 (amino acid
sequence of SEQ ID NO: 144) was generated by introducing into WT-M58 a substitution
of alanine at position 434, EU numbering.
[0509] Expression vectors for the above antibodies were constructed by the method described
in Example 1. WT-M44, WT-M58, or WT-M73 was used as H chain, while L (WT) was used
as L chain. WT-M44, WT-M58, and WT-M73 were expressed and purified by the method described
in Example 1.
Predictive assessment of the plasma retention of WT-M44, WT-M58, and WT-M73 in human
[0510] The bindings of WT-IgG1, WT-M44, WT-M58, and WT-M73 to human FcRn were assessed by
BIAcore. As shown in Table 13, the result indicates that the bindings of WT-M44, WT-M58,
and WT-M73 are greater than WT-IgG1, and about 2.7, 1.4, and 3.8 times of that of
WT-IgG1, respectively.
Table 13
|
KD(µM) |
WT-IgG1 |
1.62 |
WT-M44 |
0.59 |
WT-M58 |
1.17 |
WT-M73 |
0.42 |
[0511] As a result of assessing WT-IgG1, WT-M44, and WT-M58 for their plasma retention in
human FcRn transgenic mice, as shown in Fig. 39, WT-M58 was confirmed to have increased
retention in plasma relative to WT-IgG1 and WT-M44. Furthermore, WT-IgG1, WT-M44,
WT-M58, and WT-M73 were assessed for their plasma retention in human FcRn transgenic
mice. As shown in Fig. 40, all of WT-M44, WT-M58, and WT-M73 were confirmed to have
improved pharmacokinetics relative to WT-IgG1. The pharmacokinetics-improving effect
correlated with the binding activity to human FcRn. In particular, the plasma level
of WT-M73 at Day 28 was improved to about 16 times of that of WT-IgG1. This finding
suggests that the pharmacokinetics of antibodies with the M73 constant region in human
is also significantly enhanced when compared to antibodies with the IgG1 constant
region.
[Example 16] Effect of the novel constant regions M14 and M58 in reducing heterogeneity
in various antibodies
[0512] As described in Example 8, it was demonstrated that the heterogeneity originated
from the hinge region of IgG2 could be reduced by converting the IgG2 constant region
to M14 in the humanized anti-IL-6 receptor PM1 antibody (WT). IgG2 type antibodies
other than the humanized PM1 antibody were also tested to assess whether the heterogeneity
can be reduced by converting their constant regions into M14 or M58.
[0513] Antibodies other than the humanized PM1 antibody were: the anti IL-6 receptor antibody
F2H/L39 (the amino acid sequences of F2H/L39_VH and F2H/L39 VL as set forth in SEQ
ID NOs: 145 and 146, respectively); anti-IL-31 receptor antibody H0L0 (the amino acid
sequences of H0L0_VH and H0L0_VL as set forth in SEQ ID NOs: 147 and 148, respectively);
and anti-RANKL antibody DNS (the amino acid sequences of DNS_VH and DNS_VL as set
forth in SEQ ID NOs: 149 and 150, respectively). For each of these antibodies, antibodies
with IgG1 constant region (SEQ ID NO: 19), IgG2 constant region (SEQ ID NO: 20), or
M14 (SEQ ID NO: 24) or M58 (SEQ ID NO: 151) were generated.
[0514] The generated antibodies were assessed for heterogeneity by cation exchange chromatography
using an adequate gradient and an appropriate flow rate on a ProPac WCX-10 (Dionex)
column (mobile phase A: 20 mM sodium acetate (pH 5.0), mobile phase B: 20 mM sodium
acetate/1M NaCl (pH 5.0)). The assessment result obtained by cation exchange chromatography
(IEC) is shown in Fig. 41.
[0515] As shown in Fig. 41, conversion of the constant region from an IgG1 type into an
IgG2 type was demonstrated to increase heterogeneity not only in the humanized anti-IL-6
receptor PM1 antibody (WT), but also in the anti-IL-6 receptor antibody F2H/L39, anti-IL-31
receptor antibody H0L0, and anti-RANKL antibody DNS. In contrast, heterogeneity could
be decreased in all of these antibodies by converting their constant region into M14
or M58. Thus, it was demonstrated that, regardless of the type of antigen or antibody
variable region sequence, the heterogeneity originated from natural IgG2 could be
reduced by substituting serines for cysteines at position 131, EU numbering, in the
H-chain CH1 domain and at position 219, EU numbering, in the upper hinge of H chain.
[Example 17] Effect of the novel constant region M58 to improve the pharmacokinetics
in various antibodies
[0516] As described in Example 15, it was demonstrated that conversion of the constant region
from IgG1 into M58 in the humanized anti-IL-6 receptor PM1 antibody (WT) improved
the binding to human FcRn and pharmacokinetics in human FcRn transgenic mice. So,
IgG1 type antibodies other than the humanized PM1 antibody were also tested to assess
whether their pharmacokinetics can be improved by converting their constant region
into M58.
[0517] Antibodies other than the humanized PM1 antibody (WT) were the anti-IL-31 receptor
antibody H0L0 (the amino acid sequences of H0L0_VH and H0L0_VL as set forth in SEQ
ID NOs: 147 and 148, respectively) and anti-RANKL antibody DNS (the amino acid sequences
of DNS_VH and DNS_VL as set forth in SEQ ID NOs: 149 and 150, respectively). For each
of these antibodies, antibodies with IgG1 constant region (SEQ ID NO: 19) or M58 (SEQ
ID NO: 151) were generated, and assessed for their binding to human FcRn by the method
described in Example 14. The result is shown in Table 14.
Table 14
|
|
KD (µM) |
|
|
WT |
H0L0 |
DNS |
IgG1 |
1.42 |
1.07 |
1.36 |
M58 |
1.03 |
0.91 |
1.03 |
[0518] As shown in Table 14, it was demonstrated that as a result of conversion of the constant
region from the IgG1 type to M58, as with anti-IL-6 receptor antibody WT, the bindings
of both the anti-IL-31 receptor antibody H0L0 and anti-RANKL antibody DNS to human
FcRn were improved. This suggests the possibility that regardless of the type of antigen
or sequence of antibody variable region, the pharmacokinetics in human is improved
by converting the constant region from the IgG1 type to M58.
[Example 18] Effect of cysteine in the CH1 domain on heterogeneity and stability
[0519] As described in Example 8, cysteines in the hinge region and CH1 domain of IgG2 were
substituted to decrease the heterogeneity of natural IgG2. Assessment of various altered
antibodies revealed that heterogeneity could be reduced without decreasing stability
by using SKSC (SEQ ID NO: 154). SKSC (SEQ ID NO: 154) is an altered constant region
obtained by substituting serine for cysteine at position 131 and lysine for arginine
at position 133, EU numbering, in the H-chain CH1 domain, and serine for cysteine
at position 219, EU numbering, in the H-chain upper hinge of the wild type IgG2 constant
region sequence.
[0520] Meanwhile, another possible method for decreasing heterogeneity is a single substitution
of serine for cysteine at position 219, or serine for cysteine at position 220, EU
numbering, in the H-chain upper hinge. The altered IgG2 constant region SC (SEQ ID
NO: 155) was prepared by substituting serine for cysteine at position 219 and CS (SEQ
ID NO: 156) was prepared by substituting serine for cysteine at position 220, EU numbering,
in IgG2. WT-SC (SEQ ID NO: 157) and WT-CS (SEQ ID NO: 158) were prepared to have SC
and CS, respectively, and compared with WT-IgG1, WT-IgG2, WT-SKSC, and WT-M58 in terms
of heterogeneity and thermal stability. Furthermore, F2H/L39-IgG1, F2H/L39-IgG2, F2H/L39-SC,
F2H/L39-CS, F2H/L39-SKSC, and F2H/L39-M14, which have the constant region of IgG1
(SEQ ID NO: 19), IgG2 (SEQ ID NO: 20), SC (SEQ ID NO: 155), CS (SEQ ID NO: 156), SKSC
(SEQ ID NO: 154), or M14 (SEQ ID NO: 24), respectively, were prepared from F2H/L39
(the amino acid sequences of F2H/L39_VH and F2H/L39_VL as set forth in SEQ ID NOs:
145 and 146, respectively), which is an anti IL-6 receptor antibody different from
WT. The antibodies were compared with regard to heterogeneity and stability.
[0521] WT-IgG1, WT-IgG2, WT-SC, WT-CS, WT-SKSC, WT-M58, F2H/L39-IgG1, F2H/L39-IgG2, F2H/L39-SC,
F2H/L39-CS, F2H/L39-SKSC, and F2H/L39-M14 were assessed for heterogeneity by cation
exchange chromatography using an adequate gradient and an appropriate flow rate on
a ProPac WCX-10 (Dionex) column (mobile phase A: 20 mM sodium acetate (pH 5.0), mobile
phase B: 20 mM sodium acetate/1M NaCl (pH 5.0)). The assessment result obtained by
cation exchange chromatography is shown in Fig. 42.
[0522] As shown in Fig.42, conversion of the constant region from an IgG1 type to an IgG2
type was demonstrated to increase heterogeneity in both WT and F2H/L39. In contrast,
heterogeneity was significantly decreased by converting the constant region into SKSC
and M14 or M58. Meanwhile, conversion of the constant region into SC significantly
decreased heterogeneity, as in the case of SKSC. However, conversion into CS did not
sufficiently improve heterogeneity.
[0523] In addition to low heterogeneity, high stability is generally desired when preparing
stable formulations in development of antibody pharmaceuticals. Thus, to assess stability,
the midpoint temperature of thermal denaturation (Tm value) was determined by differential
scanning calorimetry (DSC) (VP-DSC; Microcal). The midpoint temperature of thermal
denaturation (Tm value) serves as an indicator of stability. In order to prepare stable
formulations as pharmaceutical agents, a higher midpoint temperature of thermal denaturation
(Tm value) is preferred (
J. Pharm. Sci. 2008 Apr;97(4):1414-26). WT-IgG1, WT-IgG2, WT-SC, WT-CS, WT-SKSC, and WT-M58 were dialyzed (EasySEP; TOMY)
against a solution of 20 mM sodium acetate, 150 mM NaCl, pH 6.0. DSC measurement was
carried out at a heating rate of 1°C/min in a range of 40 to 100°C, and at a protein
concentration of about 0.1 mg/ml. The denaturation curves obtained by DSC are shown
in Fig. 43. The Tm values of the Fab domains are listed in Table 15 below.
Table 15
|
Tm/°C |
WT-IgG1 |
94.8 |
WT-IgG2 |
93.9 |
WT-SC |
86.7 |
WT-CS |
86.4 |
WT-SKSC |
93.7 |
WT-M58 |
93.7 |
[0524] The Tm values of WT-IgG1 and WT-IgG2 were almost the same (about 94°C; Tm of IgG2
was about 1°C lower). Meawhile, the Tm values of WT-SC and WT-CS were about 86°C,
and thus significantly lower than those of WT-IgG1 and WT-IgG2. On the other hand,
the Tm values of WT-M58 and WT-SKSC were about 94°C, and comparable to those of WT-IgG1
and WT-IgG2. This suggests that WT-SC and WT-CS are markedly unstable as compared
to IgG2, and thus, WT-SKSC and WT-M58, both of which also comprise substituion of
serine for cysteine in the CH1 domain, are preferred in the development of antibody
pharmaceuticals-The reason for the significant decrease of Tm in WT-SC and WT-CS relative
to IgG2 is thought to be differences in the disulfide-bonding pattern between WT-SC
or WT-CS and IgG2.
[0525] Furthermore, comparison of DSC denaturation curves showed that WT-IgG1, WT-SKSC,
and WT-M58 each gave a sharp and single denaturation peak for the Fab domain. In contrast,
WT-SC and WT-CS each gave a broader denaturation peak for the Fab domain. WT-IgG2
also gave a shoulder peak on the lower temperature side of the Fab domain denaturation
peak. In general, it is considered that a single component gives a sharp DSC denaturation
peak, and when two or more components with different Tm values (namely, heterogeneity)
are present, the denaturation peak becomes broader. Specifically, the above-described
result suggests the possibility that each of WT-IgG2, WT-SC, and WT-CS contains two
or more components, and thus the natural-IgG2 heterogeneity has not been sufficiently
reduced in WT-SC and WT-CS. This finding suggests that not only cysteines in the hinge
region but also those in the CH1 domain are involved in the wild type-IgG2 heterogeneity,
and it is necessary to alter not only cysteines in the hinge region but also those
in the CH1 domain to decrease the DSC heterogeneity. Furthermore, as described above,
stability comparable to that of wild type IgG2 can be acheived only when cysteines
in both the hinge region and CH1 domain are substituted.
[0526] The above finding suggests that from the perspective of heterogeneity and stability,
SC and CS, which are constant regions introduced with serine substitution for only
the hinge region cysteine, are insufficient as constant regions to decrease heterogeneity
originated from the hinge region of IgG2. It was thus discovered that the heterogeneity
could be significantly decreased while maintaining an IgG2-equivalent stability, only
when the cysteine at position 131, EU numbering, in the CH1 domain was substituted
with serine in addition to cysteine at hinge region. Such constant regions include
M14, M31, M58, and M73 described above. In particular, M58 and M73 are stable and
less heterogeneous, and exhibit improved pharmacokinetics, and therefore are expected
to be very useful as constant regions for antibody pharmaceuticals.
[Example 19] Generation of fully humanized anti-IL-6 receptor antibodies with improved
PK/PD
[0527] To generate a fully humanized anti-IL-6 receptor antibody with improved PK/PD, the
molecules described below were created by altering TOCILIZUMAB (H chain, WT-IgG1 (SEQ
ID NO: 15); L chain, WT (SEQ ID NO: 105).
[0528] To improve the ka of F2H-IgG1, substitutions of valine for tryptophan at position
35, phenylalanine for tyrosine at position 50, and threonine for serine at position
62, which are the affinity enhancing substitution obtained in Example 2, were carried
out. Furthermore, to lower pI without increasing immunogenicity risk, substitutions
of valine for tyrosine at position 102, glutamic acid for glutamine at position 105,
and threonine for isoleucine at position 107 were carried out, and conversion of the
constant region from an IgG1 type to an M83 type was carried out and generated VH5-M83
(amino acid sequence of SEQ ID NO: 139). In addition, to improve the ka of L39, VL5-kappa
(amino acid sequence of SEQ ID NO: 181) was prepared and it comprises a substitution
of glutamine for glutamic acid at position 27. Furthermore, TOCTLIZUMAB variants were
prepared by combining two or more of the mutations in variable and constant regions
described in the above examples and newly discovered mutations. The following fully
humanized IL-6 receptor antibodies were discovered using various screening tests:
Fv3-M73 (H chain, VH4-M73, SEQ ID NO: 182; L chain, VL1-kappa, SEQ ID NO: 183), Fv4-M73
(H chain, VH3-M73, SEQ ID NO: 180; L chain, VL3-kappa, SEQ ID NO: 181), and Fv5-M83
(H chain, VH5-M83, SEQ ID NO: 139; L chain, VL5-kappa, SEQ ID NO: 138).
[0529] The affinities of prepared Fv3-M73, Fv4-M73, and Fv5-M83 against IL-6 receptor were
compared to that of TOCILIZUMAB (see Reference Example for method). The affinities
of these anti- IL-6 receptor antibodies determined are shown in Table 16. Furthermore,
their BaF/gp130-neutralizing activities were compared to those of TOCILIZUMAB and
the control (the known high affinity anti-IL-6 receptor antibody described in Reference
Example, and VQ8F11-21 hIgG1 described in
US 2007/0280945; see Reference Example for method). The results obtained by determining the biological
activities of these antibodies using BaF/gp130 are shown in Fig. 44 (TOCILIZUMAB,
the control, and Fv5-M83 with a final IL-6 concentration of 300 ng/ml) and Fig. 45
(TOCILIZUMAB, Fv3-M73, and Fv4-M73 with a final IL-6 concentration of 30 ng/ml). As
shown in Table 16, Fv3-M73 and Fv4-M73 have about two to three times higher affinity
than TOCILIZUMAB, while Fv5-M83 exhibits about 100 times higher affinity than TOCILIZUMAB
(since it was difficult to measure the affinity of Fv5-M83, instead the affinity was
determined using Fv5-IgG1, which has an IgG1-type constant region; the constant region
is generally thought to have no effect on affinity). As shown in Fig. 45, Fv3-M73
and Fv4-M73 exhibit slightly stronger activities than TOCILIZUMAB. As shown in Fig.
44, Fv5-M83 has a very strong activity, which is more than 100 times greater than
that of TOCILIZUMAB in terms of 50% inhibitory concentration. Fv5-M83 also exhibits
about 10 times higher neutralizing activity in terms of 50% inhibitory concentration
than the control (the known high-affinity anti-IL-6 receptor antibody).
Table 16
|
ka(1/Ms) |
kd(1/s) |
KD(M) |
TOCILIZUMAB |
4.0E+05 |
1.1E-03 |
2.7E-09 |
Fv3-M73 |
8.5E+05 |
8.7E-04 |
1.0E-09 |
Fv4-M73 |
7.5E+05 |
1.0E-03 |
1.4E-09 |
Fv5-M83 |
1.1 E+06 |
2.8E-05 |
2.5E-11 |
[0530] The isoelectric points of TOCILIZUMAB, the control, Fv3-M73, Fv4-M73, and Fv5-M83
were determined by isoelectric focusing using a method known to those skilled in the
art. The result showed that the isoelectric point was about 9.3 for TOCILIZUMAB; about
8.4 to 8.5 for the control; about 5.7 to 5.8 for Fv3-M73; about 5.6 to 5.7 for Fv4-M73;
and 5.4 to 5.5 for Fv5-M83. Thus, each antibody had a significantly lowered isoelectric
point when compared to TOCILIZUMAB and the control. Furthermore, the theoretical isoelectric
point of the variable regions VH/VL was calculated by GENETYX (GENETYX CORPORATION).
The result showed that the theoretical isoelectric point was 9.20 for TOCILIZUMAB;
7.79 for the control; 5.49 for Fv3-M73; 5.01 for Fv4-M73; and 4.27 for Fv5-M83. Thus,
each antibody had a significantly lowered isoelectric point when compared to TOCILIZUMAB
and the control. Accordingly, the pharmacokinetics of Fv3-M73, Fv4-M73, and Fv5-M83
was thought to be improved when compared to TOCILIZUMAB and the control.
[0531] T-cell epitopes in the variable region sequence of TOCILIZUMAB, Fv3-M73, Fv4-M73,
or Fv5-M83 were analyzed using TEPITOPE (
Methods. 2004 Dec;34(4):468-75). As a result, TOCILIZUMAB was predicted to have T-cell epitopes, of which many could
bind to HLA. In contrast, the number of sequences that were predicted to bind to T-cell
epitopes was significantly reduced in Fv3-M73, Fv4-M73, and Fv5-M83. In addition,
the framework of Fv3-M73, Fv4-M73, or Fv5-M83 has no mouse sequence and is thus fully
humanized. These suggest the possibility that immunogenicity risk is significantly
reduced in Fv3-M73, Fv4-M73, and Fv5-M83 when compared to TOCILIZUMAB.
[Example 20] PK/PD test of fully humanized anti-IL-6 receptor antibodies in monkeys
[0532] Each of TOCILIZUMAB, the control, Fv3-M73, Fv4-M73, and Fv5-M83 was intravenously
administered once at a dose of 1 mg/kg to cynomolgus monkeys to assess the time courses
of their plasma concentrations (see Reference Example for method). The plasma concentration
time courses of TOCILIZUMAB, Fv3-M73, Fv4-M73, and Fv5-M83 after intravenous administration
are shown in Fig. 46. The result showed that each of Fv3-M73, Fv4-M73, and Fv5-M83
exhibited significantly improved plasma retention in cynomolgus monkeys when compared
to TOCILIZUMAB and the control. Of them, Fv3-M73 and Fv4-M73 exhibited substantially
improved plasma retention when compared to TOCILIZUMAB.
[0533] The efficacy of each antibody to neutralize membrane-bound cynomolgus monkey IL-6
receptor was assessed. Cynomolgus monkey IL-6 was administered subcutaneously in the
lower back at 5 µg/kg every day from Day 6 to Day 18 after antibody administration
(Day 3 to Day 10 for TOCILIZUMAB), and the CRP concentration in each animal was determined
24 hours later (see Reference Example for method). The time course of CRP concentration
after administration of each antibody is shown in Fig. 47. To assess the efficacy
of each antibody to neutralize soluble Cynomolgus monkey IL-6 receptor, the plasma
concentration of free soluble cynomolgus monkey IL-6 receptor in the cynomolgus monkeys
was determined and percentage of soluble IL-6 receptor neutralization were calculated
(see Reference Example for method). The time course of percentage of soluble IL-6
receptor neutralization after administration of each antibody is shown in Fig. 48.
[0534] Each of Fv3-M73, Fv4-M73, and Fv5-M83 neutralized membrane-bound cynomolgus monkey
IL-6 receptor in a more sustainable way, and suppressed the increase of GRP over a
longer period when compared to TOCILIZUMAB and the control (the known high-affinity
anti-IL-6 receptor antibody). Furthermore, each of Fv3-M73, Fv4-M73, and Fv5-M83 neutralized
soluble cynomolgus monkey IL-6 receptor in a more sustainable way, and suppressed
the increase of free soluble cynomolgus monkey IL-6 receptor over a longer period
when compared to TOCILIZUMAB and the control. These findings demonstrate that all
of Fv3-M73, Fv4-M73, and Fv5-M83 are superior in sustaining the neutralization of
membrane-bound and soluble IL-6 receptors than TOCILIZUMAB and the control. Of them,
Fv3-M73 and Fv4-M73 are remarkably superior in sustaining the neutralization. Meanwhile,
Fv5-M83 suppressed CRP and free soluble cynomolgus monkey IL-6 receptor more strongly
than Fv3-M73 and Fv4-M73. Thus, Fv5-M83 is considered to be stronger than Fv3-M73,
Fv4-M73, and the control (the known high-affinity anti-IL-6 receptor antibody) in
neutralizing membrane-bound and soluble IL-6 receptors. It was considered that results
in
in vivo of Cynomolgus monkeys reflect the stronger affinity of Fv5-M83 for IL-6 receptor
and stronger biological activity of Fv5-M83 in the BaF/gp130 assay system relative
to the control.
[0535] These findings suggest that Fv3-M73 and Fv4-M73 are highly superior in sustaining
their activities as an anti-IL-6 receptor-neutralizing antibody when compared to TOCILIZUMAB
and the control, and thus enable to significantly reduce the dosage and frequency
of administration. Furthermore, Fv5-M83 was demonstrated to be remarkably superior
in terms of the strength of activity as an anti-IL-6 receptor-neutralizing antibody
as well as sustaining their activity. Thus, Fv3-M73, Fv4-M73, and Fv5-M83 are expected
to be useful as pharmaceutical IL-6 antagonists.
[Reference Example]
Preparation of soluble recombinant Cynomolgus monkey IL-6 receptor (cIL-6R)
[0536] Oligo-DNA primers were prepared based on the disclosed gene sequence for Rhesus monkey
IL-6 receptor (
Birney et al., Ensembl 2006, Nucleic Acids Res. 2006 Jan 1 ;34 (Database issue):D556-61). A DNA fragment encoding the whole cynomolgus monkey IL-6 receptor gene was prepared
by PCR using the primers, and as a template, cDNA prepared from the pancreas of cynomolgus
monkey. The resulting DNA fragment was inserted into an animal cell expression vector,
and a stable expression CHO line (cyno.sIL-6R-producing CHO cell line) was prepared
using the vector. The culture medium of cyno.sIL-6R-producing CHO cells was purified
using a HisTrap column (GE Healthcare Bioscience) and then concentrated with Amicon
Ultra-15 Ultracel-10k (Millipore). A final purified sample of soluble cynomolgus monkey
IL-6 receptor (hereinafter cIL-6R) was obtained through further purification on a
Superdex200pg16/60 gel filtration column (GE Healthcare Bioscience).
Preparation of Recombinant cynomolgus monkey IL-6 (cIL-6)
[0537] Cynomolgus monkey IL-6 was prepared by the procedure described below. The nucleotide
sequence encoding 212 amino acids deposited under SWISSPROT Accession No. P79341 was
prepared and cloned into an animal cell expression vector. The resulting vector was
introduced into CHO cells to prepare a stable expression cell line (cyno.IL-6-producing
CHO cell line). The culture medium of cyno.IL-6-producing CHO cells was purified using
a SP-Sepharose/FF column (GE Healthcare Bioscience) and then concentrated with Amicon
Ultra-15 Ultracel-5k (Millipore). A final purified sample of cynomolgus monkey IL-6
(hereinafter cIL-6) was obtained through further purification on a Superdex75pg26/60
gel filtration column (GE Healthcare Bioscience), followed by concentration with Amicon
Ultra-15 Ultracel-5k (Millipore).
Preparation of a known high-affinity anti-IL-6 receptor antibody
[0538] An animal cell expression vector was constructed to express VQ8F11-21 hIgG1, a known
high-affnity anti-IL-6 receptor antibody. VQ8F11-21 hIgG1 is described in
US 2007/0280945 A1 (
US 2007/0280945 A1; the amino acid sequences of H chain and L chain as set forth in SEQ ID NOs: 19 and
27, respectively). The antibody variable region was constructed by PCR using a combination
of synthetic oligo DNAs (assembly PCR). IgG1 was used as the constant region. The
antibody variable and constant regions were combined together by assembly PCR, and
then inserted into an animal cell expression vector to construct expression vectors
for the H chain and L chain of interest. The nucleotide sequences of the resulting
expression vectors were determined by a method known to those skilled in the art.
[0539] The high-affinity anti-IL-6 receptor antibody (hereinafter abbreviated as "control")
was expressed and purified using the constructed expression vectors by the method
described in Example 1.
Biacore-based analysis of binding to IL-6 receptor
[0540] Antigen-antibody reaction kinetics was analyzed using Biacore T100 (GE Healthcare).
The SR344-antibody interaction was measured by immobilizing appropriate amounts of
anti-IgG (γ-chain specific) F(ab')
2 onto a sensor chip by amine coupling method, binding antibodies of interest onto
the chip at pH7.4, and then running IL-6 receptor SR344 adjusted to be various concentrations
at pH7.4 over the chip as an analyte. All measurements were carried out at 37°C. The
kinetic parameters, association rate constant k
a (1/Ms) and dissociation rate constant k
d (1/s) were calculated from the sensorgrams obtained by measurement. Then, K
D (M) was determined based on the rate constants. The respective parameters were determined
using Biacore T100 Evaluation Software (GE Healthcare).
PK/PD test to determine the plasma concentrations of antibodies, CRP, and free soluble
IL-6 receptor in monkeys
[0541] The plasma concentrations in cynomolgus monkeys were determined by ELISA using a
method known to those skilled in the art.
[0542] The concentration of CRP was determined with an automated analyzer (TBA-120FR; Toshiba
Medical Systems Co.) using Cias R CRP (KANTO CHEMICAL CO., INC.).
[0543] The plasma concentration of free soluble cynomolgus monkey IL-6 receptor in cynomolgus
monkeys was determined by the procedure described below. All IgG antibodies (cynomolgus
monkey IgG, anti-human IL-6 receptor antibody, and anti-human IL-6 receptor antibody-soluble
cynomolgus monkey IL-6 receptor complex) in the plasma were adsorbed onto Protein
A by loading 30 µl of cynomolgus monkey plasma onto an appropriate amount of rProtein
A Sepharose Fast Flow resin (GE Healthcare) dried in a 0.22-µm filter cup (Millipore).
Then, the solution in cup was spinned down using a high-speed centrifuge to collect
the solution that passed through. The solution that passed through does not contain
Protein A-bound anti-human IL-6 receptor antibody-soluble cynomolgus monkey IL-6 receptor
complex. Therefore, the concentration of free soluble IL-6 receptor can be determined
by measuring the concentration of soluble cynomolgus monkey IL-6 receptor in the solution
that passed through Protein A. The concentration of soluble cynomolgus monkey IL-6
receptor was determined using a method known to those skilled in the art for measuring
the concentrations of soluble human IL-6 receptor. Soluble cynomolgus monkey IL-6
receptor (cIL-6R) prepared as described above was used as a standard.
[0544] Then the percentage of soluble IL-6 receptor neutralization was calculated by following
formula.

Industrial Applicability